Methods to produce chimeric adeno-associated virus/bocavirus parvovirus

ABSTRACT

A method of preparing a chimeric virus comprising bocavirus capsid protein (VP) and a recombinant adeno-associated (AAV) viral genome, and isolated mutant bocavirus genomes, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.application Ser. No. 62/292,613, filed on Feb. 8, 2016 and U.S.application Ser. No. 62/453,745, filed on Feb. 2, 2017, the disclosuresof which are incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under grants AI105543and AI112803 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND

Human bocavirus1 (HBoV1), first identified in 2005 (Allander et al.,2005), belongs to genus Bocaparvovirus in the subfamily Parvovirinae ofthe Parvoviridae family (Cotmore et al., 2014). The genus Bocaparvovirusconsists of three groups of viruses, namely HBoV1-4, bovine parvovirus 1(BPV1) and minute virus of canines (MVC/CnMV) (Johnson and Qiu, 2011).HBoV1 causes respiratory tract infection in young children worldwide(Allander et al., 2007; Christensen et al., 2010; Deng et al., 2012; Donet al., 2010; Edner et al., 2011; Kantola et al., 2008; Martin et al.,2015). In vitro, the virus infects only polarized (well-differentiated)human airway epithelium cultured at an air-liquid interface (HAE-ALI)(Dijkman et al., 2009; Huang et al., 2012; Deng et al., 2013; Deng etal., 2014). An infectious clone of HBoV1 (pIHBoV1) was constructed.Transfecting the pIHBoV1 in human embryonic kidney 293 (HEK293) cellsresults in efficient replication of the HBoV1 genome and production ofHBoV1 virions, which are infectious in HAE-ALI (Huang et al., Deng etal., 2013).

The transcription profile of HBoV1 has been studied in transfection ofboth HBoV1 non-replicating and replicating double-stranded DNA (dsDNA)forms of the viral genome in HEK293 cells (Chen et al., 2010; Shen etal., 2015), as well as during HBoV1 infection of HAE-ALI (Dijkman etal., 2009; Shen et al., 2015). All the viral mRNA transcripts aregenerated from alternative processing (involving both splicing andpolyadenylation) of one HBoV1 precursor-RNA (pre-mRNA), which istranscribed from the P5 promoter (FIG. 1A) (Dijkman et al., 2009; Chenet al., 2010; Shen et al., 2015). The left side of the genome encodesnon-structural (NS) proteins, and four major NS proteins (NS1, NS2, NS3,and NS4) are expressed from alternatively spliced mRNA transcripts (Shenet al., 2015). While NS1 is critical to viral DNA replication, NS2 alsoplays a role during virus replication in infection of HAE-ALI (Shen etal., 2015). The right side of the genome encodes viral capsid proteinsfrom alternatively spliced mRNA transcripts, R6, R7, and R8 mRNAs (FIG.1A). Of note, HBoV1, like other members of the genus Bocaparvovirus,encodes a unique nonstructural protein NP1 from the middle of thegenome. NP1 is required for an efficient replication of viral DNA (Huanget al., 2012; Sun et al., 2009).

HBoV1 capsid is capable of cross-genus packaging of a genome ofrecombinant adeno-associated virus 2 (rAAV2) in HEK293 cells, whichgenerates HBoV1 capsid-pseudotyped 129 rAAV2 vector (rAAV2/HBoV1) (Yanet al., 2013). The chimeric vector can deliver a full-length cysticfibrosis transmembrane conductance regulator (CFTR) gene with a strongpromoter to cystic fibrosis (CF) HAE, with demonstrated efficacy incorrecting CFTR-dependent chloride transport (Yan et al., 2013).Therefore, the rAAV2/HBoV1-CFTR vector holds much promise for CF genetherapy. However, in the current vector production system, the packagingplasmid pHBoV1NSCap, which carries an HBoV1 non-replicating dsDNA genome(containing the P5 promotor and NS and Cap genes), was used forpackaging rAAV2/HBoV1 vector. The efficiency of the vector production ison average 10 times lower than that of the rAAV2 vector packaged by theAAV2 capsid (Yan et al., 2013). It was hypothesized that this lowerefficiency is likely due to the unnecessary expression of the HBoV1 NSgene from the packaging plasmid pHBoV1NSCap.

SUMMARY

Chimeric parvovirus vector rAAV/HBoV inherits the safety of rAAV(recombinant adeno-associated virus) and the airway tropism of HBoV(human bocavirus type 1), which provides a solution for the current lackof an efficient vector for airway gene therapy of pulmonary diseases,such as cystic fibrosis, alpha-antitrypsin deficiency, asthma, vaccines,and lung cancer.

This disclosure provides improved methods to produce chimeric AAV/BoVviruses, e.g., using a chimeric parvoviral vector (rAAV2/HBoV1) vector,in which the recombinant adeno-associated virus (rAAV) genome ispseudo-packaged by the human bocavirus 1 (HBoV1) capsid, such as oneexpressing a full-length cystic fibrosis transmembrane conductanceregulator (CFTR) gene that is capable of correcting CFTR81 dependentchloride transport in cystic fibrosis human airway epithelium.Previously, an HBoV1 nonstructural (NS) and capsid (Cap)protein-expressing plasmid, pHBoV1NSCap, was used to package arAAV2/HBoV1 vector, but yields remain low, e.g., 2×10¹¹ DNAase-resistantparticles (DRPs)/40 150 mm dishes of transfected cells (400-600DRP/cell), which is about 5% to about 10% the typical yield of rAAV2production). In order to simplify this packaging plasmid, theinvolvement of the HBoV1 nonstructural proteins in capsid proteinexpression was investigated as described below. It was found that NP1, asmall NS protein encoded by the middle open reading frame, allows forthe expression of the viral capsid proteins (VP1, VP2, and VP3), whereasthe other NS proteins (NS1, NS2, NS3, and NS4) are not necessarilyrequired. Systematic analyses of the HBoV1 mRNAs transcribed from thepHBoV1NSCap packaging plasmid and its derivatives in HEK293 cells wereperformed. By mutating the cis-elements, which function as internalpolyadenylation signals in the capsid protein-expressing mRNA, a simpleHBoV1 capsid protein gene was constructed that expresses capsid proteinsVP1, VP2 and VP3 as efficiently as pHBoV1NSCap and at similar ratios,but independently of the NP1. Thus, mechanistically, it was found thatNP1 is required for both the splicing and the read-through of theproximal polyadenylation site of the HBoV1 precursor-mRNA, both of whichare essential for the maturation of capsid protein-encoding mRNA. Thus,the present disclosure provides a unique example of how a small viralnonstructural protein facilitates the multifaceted regulation of capsidgene expression. Moreover, a function of HBoV1 NP1 in the regulation ofcapsid protein expression was identified, which allows for a betterpackaging system for rAAV/HBoV vector production.

The present invention provides a method of preparing a chimeric viruscomprising bocavirus capsid protein (VP) and a recombinantadeno-associated (AAV) viral genome. Any AAV genome may be employed,e.g., any mammalian AAV including any primate AAV, a human or non-humanprimate AAV, including a chimeric AAV genome such as one havingsynthetic or chimeric ITRs (see, e.g., U.S. Pat. No. 8,241,622, which isincorporated by reference herein), or formed from AAV DNA from two ormore different serotypes, e.g., a chimeric genome having 2 ITRs, eachITR from a different serotype or chimeric ITRs. Any bocavirus orbocavirus genome may be employed in the methods, for example, a humanbocavirus or human bocavirus genome. In one embodiment, the method isNP1 dependent, and according to one embodiment, the method comprisesproviding a mutant bocavirus, e.g., HoBV1, genome that when introducedinto cells; i) does not express one or more of NS1, NS2, NS3 or NS4; forexample, expresses none of NS1, NS2, NS3, NS4; or expresses only NS1 orNS2 or NS3 or NS4; or expresses any two of NS1, NS2, NS3, NS4, forexample, expresses NS3 and NS4; or expresses any three of NS1, NS2, NS3,NS4, for example, expresses NS2, NS3 and NS4, ii) expresses one or moreof bocavirus VP1, VP2 or VP3, for example, expresses VP1 and VP2 andVP3, or expresses VP1 and VP2, or expresses VP1 and VP3, or expressesVP2 and VP3, and iii) expresses bocavirus NP1. The method can furtherinclude introducing the mutant bocavirus genome and a rAAV vector intocells which do not express one or more of bocavirus NS1, NS2, NS3 orNS4, as defined above thereby producing bocavirus VP, bocavirus NP1 anda rAAV genome; and can further comprise collecting chimericrAAV/bocavirus. Optionally the collecting of chimeric rAAV/bocavirus canbe from the cells or the cell supernatant, for example, where the cellsare grown in culture medium.

According to one embodiment the method comprises providing one or morevectors that express one or more of bocavirus VP1, VP2 or VP3; forexample, express VP1 and VP2 and VP3, or express VP1 and VP2, or expressVP1 and VP3, or express VP2 and VP3, and express bocavirus NP1 but donot express one or more of bocavirus NS1, NS2, NS3 or NS4, for example,expresses none of NS1, NS2, NS3, NS4, or expresses only NS1 or NS2 orNS3 or NS4; or expresses any two of NS1, NS2, NS3, NS4, for example,expresses NS3 and NS4; or expresses any three of NS1, NS2, NS3, NS4, forexample, expresses NS2, NS3 and NS4. The method can further compriseexpressing the one or more vectors in cells which do not express one ormore of bocavirus NS1, NS2, NS3 or NS4, as defined above therebyproducing bocavirus VP, bocavirus NP1 and a rAAV genome; and can furthercomprise collecting chimeric rAAV/bocavirus, optionally the collectingchimeric rAAV/bocavirus can be from the cells or the cell supernatant,for example, where the cells are grown in culture medium The one or morevectors may be integrated into the genome of the cells. The bocaviruscan be a human bocavirus or the genome a human bocavirus genome.

According to the invention, the mutant bocavirus genome and a rAAVvector can be introduced into cells thereby producing bocavirus capsidprotein (VP) and bocavirus NP1, and a rAAV genome. A mutant BoV genome,e.g., a mutant human BoV genome or a non-human BoV genome, may includeone or more insertions, one or more deletions, or one or more nucleotidesubstitutions, e.g., substitutions that results in an in-frame stopcodon or removal of a splice site, or any combination thereof, in orderto inhibit or prevent expression of a functional viral protein. Thechimeric rAAV/bocavirus virus can then be collected, optionally thecollecting chimeric rAAV/bocavirus can be from the cells or the cellsupernatant, for example, where the cells are grown in culture medium.According to the invention, the one or more vectors may be integratedinto the genome of the cells. According to the invention the bocavirusmay be a human bocavirus or the genome may be a human bocavirus genome.

In one embodiment, the rAAV vector comprises an expression cassetteencoding a heterologous gene product. In one embodiment, the geneproduct encodes a therapeutic protein. In one embodiment, the rAAVgenome is a rAAV-1, rAAV-2, rAAV-3, rAAV-4, rAAV-5, rAAV-6, rAAV-7,rAAV-8 or rAAV-9 genome. In one embodiment, the gene product is a viral,bacterial, tumor, parasite, or fungal antigen. In one embodiment, thegene product is cystic fibrosis transmembrane conductance regulator, C1inhibitor gene, C1-INH gene, SERPING gene, β-globin, γ-globin, tyrosinehydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C,erythropoietin, HBoV protein, influenza virus protein, RSV protein, aneutralizing antibody or an antigen binding fragment thereof, SARS virusprotein, or a cytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, orIL-6. In one embodiment, the gene product is cystic fibrosistransmembrane conductance regulator. In one embodiment, the gene productis C1 inhibitor gene, C1-INH gene, or SERPING1 gene. In one embodiment,the DNAase-resistant particles, DRPs, of the chimeric virus which areproduced are at least about 5% greater than the DRPs produced of acorresponding chimeric virus where the bocavirus genome also expressesbocavirus NS protein, for example, that expresses all of NS1, NS2, NS3,NS4, or that expresses one or more of NS1, NS2, NS3 or NS4, or thatexpresses the one or more of NS1, NS2, NS3 or NS4 not expressed by thechimeric virus produced by the methods described herein. In oneembodiment, the yield is about 1,000 to about 10,000 DRP/cell, e.g.,about 1,500 to about 6,000 DRP/cell. The AAV genome may be stablyincorporated into the genome of the cell, e.g., via homologousrecombination or random integration.

Also provided is a method of preparing a chimeric virus comprisingbocavirus capsid protein and a recombinant adeno-associated (AAV) viralgenome, which method is NP1 dependent. The method comprises providingone or more vectors that express one or more of VP1, VP2 or VP3, forexample, express VP1 and VP2 and VP3, or express VP1 and VP2, or expressVP1 and VP3, or express VP2 and VP3, and express NP1 but do not expressone or more of NS1, NS2, NS3 or NS4, for example, expresses none of NS1,NS2, NS3, NS4; or expresses only NS1 or NS2 or NS3 or NS 4; or expressesany two of NS1, NS2, NS3, NS4, for example, expresses NS3 and NS4; orexpresses any three of NS1, NS2, NS3, NS4, for example, expresses NS2,NS3 and NS4; expressing the vectors in cells; optionally wherein thecells do not express one or more of bocavirus NS1, NS2, NS3 or NS4, asdefined above, and collecting chimeric virus. Optionally the collectingof chimeric virus can be from the cells or the cell supernatant, forexample, where the cells are grown in culture medium. In one embodiment,the rAAV vector comprises an expression cassette encoding a heterologousgene product. In one embodiment, the gene product encodes a therapeuticprotein. In one embodiment, the rAAV genome is a rAAV-1, rAAV-2, rAAV-3,rAAV-4, rAAV-5, rAAV-6, rAAV-7, rAAV-8 or rAAV-9 genome. In oneembodiment, the gene product is a viral, bacterial, tumor, parasite, orfungal antigen. In one embodiment, the gene product is cystic fibrosistransmembrane conductance regulator, C1 inhibitor gene, C1-INH gene,SERPING gene, β-globin, γ-globin, tyrosine hydroxylase,glucocerebrosidase, aryl sulfatase A, factor VIII, dystrophin, alpha1-antitrypsin, surfactant protein SP-D, SP-A or SP-C, erythropoietin,HBoV protein, influenza virus protein, RSV protein, a neutralizingantibody or an antigen binding fragment thereof, SARS virus protein, ora cytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, or IL-6. Inone embodiment, the gene product is cystic fibrosis transmembraneconductance regulator. In one embodiment, the gene product is C1inhibitor gene, C1-INH gene, or SERPING1 gene. In one embodiment, DRP ofthe chimeric virus is at least about 5% greater than a correspondingchimeric virus where the bocavirus genome also expresses bocavirus NSprotein, for example that expresses all of NS1, NS2, NS3, NS4, that alsoexpresses one or more of NS1, NS2, NS3 or NS4, or that expresses the oneor more of NS1, NS2, NS3 or NS4 not expressed by the chimeric virus ofthe invention.

Further provided is a method of preparing a chimeric virus comprisingbocavirus capsid protein and a recombinant adeno-associated (AAV) viralgenome, which method is independent of bocavirus NP1. In one embodiment,the method comprises providing a mutant bocavirus genome that whenintroduced into cells expresses one or more of bocavirus NS1, NS2, NS3or NS4, for example, that expresses only NS1 or NS2 or NS3 or NS4; orexpresses any two of NS1, NS2, NS3, NS4, for example, expresses NS3 andNS4; or expresses any three of NS1, NS2, NS3, NS4, for example,expresses NS2, NS3 and NS4, or that expresses all of NS1, NS2, NS3, NS4;or alternatively expresses none of NS1, NS2, NS3, NS4, expresses one ormore of bocavirus VP1, VP2 or VP3, for example, expresses VP1 and VP2and VP3, or expresses VP1 and VP2, or expresses VP1 and VP3, orexpresses VP2 and VP3, but does not express bocavirus NP1 or afunctional bocavirus NP1, wherein optionally the coding region for theone or more of the VP1, VP2 or VP3 is codon optimized (SEQ ID Nos. 2-3).In one embodiment, the mutant genome expresses VP1, VP2 and VP3. In oneembodiment, the method includes providing a mutant bocavirus genome thatwhen introduced into cells does not express any of bocavirus NS1, NS2,NS3 or NS4, expresses one or more of bocavirus VP1, VP2 or VP3, forexample, expresses VP1 and VP2 and VP3, or expresses VP1 and VP2, orexpresses VP1 and VP3, or expresses VP2 and VP3, but does not expressbocavirus NP1 or a functional bocavirus NP1, wherein optionally thecoding region for the one or more of the VP1, VP2 or VP3 is codonoptimized (SEQ ID Nos. 2-3). In one embodiment, the method includesproviding one or more vectors that when introduced to cells express oneor more of bocavirus VP1, VP2 or VP3, but do not express bocavirus NP1or a functional bocavirus NP1 or any of NS1, NS2, NS3 or NS4, whereinoptionally the coding region for the one or more of the VP1, VP2 or VP3is codon optimized (SEQ ID Nos. 2-3). Optionally or additionally, themutant genome and a rAAV vector are introduced into cells that do notexpress NP1 in trans, for example, the method can further compriseintroducing the mutant bocavirus genome and a rAAV vector into cellsthat do not express bocavirus NP1 in trans, thereby producing bocavirusnonstructural proteins NS1, NS2, NS3 or NS4, capsid proteins and a rAAVgenome. The method can further comprise collecting the chimeric virus orchimeric rAAV/bocavirus, and optionally the collecting can be from thecells or the cell supernatant, for example, where the cells are grown inculture medium. In one embodiment, the promoter that expresses bocavirusproteins is not a bocavirus promoter, e.g., not a HBoV promoter. Thebocavirus can be human bocavirus. In one embodiment, the rAAV vectorcomprises an expression cassette encoding a heterologous gene product.In one embodiment, the rAAV genome is a rAAV-1, rAAV-2, rAAV-3, rAAV-4,rAAV-5, rAAV-6, rAAV-7, rAAV-8 or rAAV-9 genome wherein the gene productencodes a therapeutic protein. In one embodiment, the gene product is aviral, bacterial, tumor, parasite, or fungal antigen. In one embodiment,the gene product is cystic fibrosis transmembrane conductance regulator,C1 inhibitor gene, C1-INH gene, SERPING1 gene, β-globin, γ-globin,tyrosine hydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C,erythropoietin, HBoV protein, influenza virus protein, RSV protein, aneutralizing antibody or an antigen binding fragment thereof, SARS virusprotein, or a cytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, orIL-8. In one embodiment, the gene product is cystic fibrosistransmembrane conductance regulator. In one embodiment, the gene productis C1 inhibitor gene, C1-INH gene, or SERPING1 gene. In one embodiment,the DRP of the chimeric virus is at least about 5% greater than acorresponding chimeric virus with a bocavirus genome that also expressesbocavirus NS protein, for example that expresses all of NS1, NS2, NS3NS4, that also expresses one or more of NS1, NS2, NS3 or NS4, or thatexpresses the one or more of NS1, NS2, NS3 or NS4 not expressed by thechimeric virus of the invention. According to one embodiment of themethod of the invention neither the cells nor the mutant genome providesfor expression of any of bocavirus NS1, NS2, NS3 or NS4.

In one embodiment, the invention also provides a method of preparing achimeric virus comprising bocavirus capsid protein and a recombinantadeno-associated (AAV) viral genome which method is independent ofbocavirus NP1. In one embodiment, the method comprises providing one ormore vectors that when introduced to cells express one or more of NS1,NS2, NS3 or NS4, for example, that expresses only NS1 or NS2 or NS3 orNS 4; or expresses any two of NS1, NS2, NS3, NS4, for example, expressesNS3 and NS4; or expresses any three of NS1, NS2, NS3, NS4, for example,expresses NS2, NS3 and NS4, or that expresses all of NS1, NS2, NS3, NS4,or alternatively do not express any of NS1, NS2, NS3, NS4; express oneor more of bocavirus VP1, VP2 or VP3, for example, express VP1 and VP2and VP3, or express VP1 and VP2, or express VP1 and VP3, or express VP2and VP3, but do not express bocavirus NP1 or a functional bocavirus NP1,wherein the coding region for the one or more of the bocavirus VP1, VP2or VP3 may be codon optimized. In one embodiment, the method includesproviding one or more vectors that when introduced to cells express oneor more of bocavirus VP1, VP2 or VP3, but do not express bocavirus NP1or a functional bocavirus NP1 or any of NS1, NS2, NS3 or NS4. In oneembodiment, the one or more vectors express VP1, VP2 and VP3. In oneembodiment, the coding region for the one or more of the bocavirus VP1,VP2 or VP3 is codon optimized. Those vectors and a rAAV vector areexpressed in cells that do not express NP1 in trans thereby, producingbocavirus capsid protein and a rAAV genome; and chimeric virus iscollected. For example the method can further comprise expressing theone or more vectors and a rAAV vector in cells that do not express NP1in trans, thereby producing bocavirus nonstructural proteins NS1, NS2,NS3 or NS4 capsid protein and a rAAV genome; and collecting chimericrAAV/bocavirus, optionally the collecting chimeric virus can becollecting from the cells or the cell supernatant, for example where thecells are grown in culture medium. The method can further comprise thatthe one or more vectors are integrated into the genome of the cell andcan additionally or alternatively comprise that neither the cells northe one or more vectors provides for expression of any of bocavirus NS1,NS2, NS3 or NS4.

According to some embodiments of the invention, the promoter thatexpresses bocavirus proteins is not a bocavirus promoter.

According to a further aspect of the invention there is provided anisolated mutant bocavirus genome that when introduced to cells does notexpress one or more of bocavirus NS1, NS2, NS3 or NS4, for example,expresses none of NS1, NS2, NS3, NS4; or expresses only NS1 or NS2 orNS3 or NS 4; or expresses any two of NS1, NS2, NS3, NS4, for example,expresses NS3 and NS4; or expresses any three of NS1, NS2, NS3, NS4, forexample, expresses NS2, NS3 and NS4; expresses bocavirus VP1, VP2 andVP3; and expresses bocavirus NP1. Optionally the promoter that expressesbocavirus proteins is not a bocavirus promoter.

According to a further aspect of the invention there is provided anisolated mutant bocavirus genome that when introduced to cells i)expresses one or more of bocavirus NS1, NS2, NS3 or NS4, for example,that expresses only NS1 or NS2 or NS3 or NS 4; or expresses any two ofNS1, NS2, NS3, NS4, for example, expresses NS3 and NS4; or expresses anythree of NS1, NS2, NS3, NS4, for example, expresses NS2, NS3 and NS4, orthat expresses all of NS1, NS2, NS3, NS4, ii) expresses bocavirus VP1,VP2 and VP3; and ii) does not express bocavirus NP1 or a functionalbocavirus NP1. Optionally the promoter that expresses bocavirus proteinsis not a bocavirus promoter.

According to a further aspect of the invention there is provided anisolated mutant bocavirus genome that when introduced to cells i) doesnot express any of bocavirus NS1, NS2, NS3 or NS4; ii) expressesbocavirus VP1, VP2 and VP3; and iii) does not express bocavirus NP1 or afunctional bocavirus NP1. Optionally the promoter that expressesbocavirus proteins is not a bocavirus promoter.

According to a further aspect of the invention there is provided anisolated mutant bocavirus genome and bocavirus protein expressionvector(s). A mutant BoV genome, e.g., a mutant human BoV genome or anon-human BoV genome, may include one or more insertions, one or moredeletions, or one or more nucleotide substitutions, e.g., substitutionsthat results in an in-frame stop codon or removal of a splice site, orany combination thereof, in order to inhibit or prevent expression of afunctional viral protein.

In one embodiment, there is provided an expression vector comprising acodon optimized open reading frame for bocavirus NP1, or bocavirus VP1,VP2 or VP3, or a portion thereof. The open reading frame for NP1 canencode a protein with at least 80%, 85%, 90%, 95%, 98% or more identityto the protein encoded by SEQ ID NO:1, that is, NP1 useful in themethods may be a protein with at least 80%, 85%, 90%, 95%, 98% or moreidentity to the protein encoded by SEQ ID NO:1. The open reading framefor bocavirus VP1, VP2 and VP3 can encode proteins with at least 80%,85%, 90%, 95%, 98% or more identity to the protein encoded by any one ofSEQ ID Nos. 2-3, e.g., bocavirus VP1, VP2 and VP3 useful in the methodsmay be a protein with at least 80%, 85%, 90%, 95%, 98% or more identityto the protein encoded by any one of SEQ ID Nos. 2-3. Coding regions forNP1 and capsid proteins from AAVs, e.g., other than AAV2, may likewiseby codon optimized. According to one embodiment the expression vectorcomprises a heterologous promoter linked to the open reading frame.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-C. Genetic map of HBoV1 and the probes used. A-B) Theexpression profile of HBoV1 mRNAs and their encoded proteins are shownwith transcription landmarks and boxed open reading frames (ORFs). Thenumbers are nucleotide (nt) numbers of the HBoV1 full-length genome(GenBank accession no.:JQ923422). Major species of HBoV1 mRNAtranscripts that are alternatively processed are shown with their sizesin kilobases (kb) (minus a poly A tail of about 150 nts), which wereconcluded from multiple studies in HBoV1 DNA transfection of HEK293cells and in HBoV1 infection of polarized human airway epithelium(Dijkman et al., 2009; Chen et al., 2010; Shen et al., 2015). Viralproteins detected in both transfected and infected cells are shown sideby side at the right. P5, P5 promoter; D, 5′ splice donor site; A, 3′splice acceptor site; (pA)p and (pA)d, proximal and distalpolyadenylation sites, respectively; LEH/REH, left/right end hairpin. C)Probes used for Northern blot analysis are depicted with the nucleotidenumbers. Probes used for RNAse protection assay (RPA) are diagrammedwith the sizes of their detected bands.

FIG. 2. DNA sequence of silent mutations in the (pA)p sites. Nucleotidesequences between the VP1 and VP3 start codons were silently mutated toeliminate potentially used (cryptic) polyadenylation signals (PAS:AAUAAA), but still retain the same coding sequence as the wild type. Themutated sequence is shown with locations of the 5 PASs (dashedunderline). The VP1 start codon was mutated from ATG to ACG, togetherwith a weak Kozak signal (GTT AAG), and retained VP2 start codon GTG.

FIGS. 3A-B. VP2 is translated from a non-canonical translationinitiation codon. A) Diagrams of pHBoV1NSCap-based mutants. The parentpHBoV1NSCap plasmid is diagrammed with transcription, splicing, andpolyadenylation units shown, together with the mutants that carryvarious mutations at the GUG and GCU codons (nt 3,422-3,427) of theHBoV1 genome, as shown. B) Western blot analysis of capsid proteins.HEK293 cells were transfected with plasmids as indicated. The lysates oftransfected cells were analyzed by Western blotting using an anti-VPantibody, and was reprobed with anti-β-actin. The identities of thedetected bands are indicated at the left of the blot. Arrow shows thenovel VP2 protein, which was denoted previously by an asterisk (*) (Shenet al., 2015). Control, without transfection.

FIGS. 4A-D. HBoV1 VP cDNA constructs did not express capsid proteins. A)Diagrams of HBoV1 VP cDNA constructs. pHBoV1NSCap is diagramed withtranscription, splicing, and polyadenylation units shown. pCMVCap1-3 andpCMVR6-8cDNA that contain various sequences of the 5′ untranslationalregion (UTR) and 3′UTR and the VP1/2/3 ORF are diagramed with thejunction of the 5′ and 3′ splice sites or the heterogeneous Epo intron 4(Epo In4) as shown. B) Western blot analysis of capsid proteins. HEK293cells were transfected with plasmids as indicated. The lysates oftransfected cells were analyzed by Western blotting using an anti-VPantibody, and were reprobed with anti-β-actin. The lysates were alsoanalyzed by Western blotting using an anti-HA antibody for detecting theC-terminal HA-tagged mCherry. The identities of the detected bands areindicated at the left of the blot. C) Northern blot analysis ofcytoplasmic VP mRNAs. HEK293 cells were transfected with plasmids asindicated. Cytoplasmic RNA prepared from each transfection was analyzedby Northern blotting using the Cap probe, which specifically detects VPmRNA (FIG. 1B). EB stained 18S rRNA bands are shown, and the VP mRNAbands are indicated. Asterisk (*) denotes various NS-encoding mRNAs. ARNA ladder (M) was used as a size marker. D) Quantification of VP mRNAexpression. The bands of VP mRNA in each lane of panel C were quantifiedand normalized to the level of 18S rRNA. The signal intensity of the VPmRNA band in lane 1 was arbitrally set as 100%. Relative intensity wascalculated for the bands in other lanes. Means and standard deviationswere calculated from at least three independent experiments.

FIGS. 5A-D. Knockout of NP1 abolished VP mRNA production and capsidprotein expression. A) Diagrams of HBoV1 NSCap gene constructs.pHBoV1NSCap and its derivatives are diagramed with replacement of theCMV promoter and bGHpA signal and knockout of the NS1 and NP1 as shown.B) Western blot analysis of capsid proteins. HEK293 cells weretransfected with plasmids as indicated. Cell lysates were analyzed byWestern blotting using an anti-VP antibody. The blot was reprobed withanti-β-actin. The lysates were also analyzed by Western blotting usinganti-NP1 and anti-HA antibodies. The identities of detected proteins areshown at the left of the blot. C) Northern blot analysis of cytoplasmicVP mRNAs. HEK293 cells were transfected with plasmids as indicated.Cells were harvested and extracted for cytoplasmic RNA at 2 dayspost-transfection. The cytoplasmic RNA samples were analyzed by Northernblotting using the Cap probe. EB-stained 18S rRNA and detected VP mRNAare indicated. Asterisk (*) denotes various NS-encoding mRNAs. D)Western blot analysis of NS proteins. The same lysates prepared forpanel B were analyzed by Western blotting using an anti-NS1C antibody.The identities of detected proteins are shown at the left of the blot.

FIGS. 6A-H. NP1 complementation rescued VP mRNA production and capsidproteins expression. A) Diagrams of HBoV1 NSCap constructs. pCMVNS*Capand pCMVNS*(NP*)Cap are diagrammed, along with the pOpt-NP1 plasmid thatexpresses NP1 from a codon-optimized NP1 ORF (SEQ ID NO:1). (B) RPAanalysis of viral RNAs spliced at the A1. D2 and A3 splice sites. Ten μgof total RNA isolated at 2 days post-transfection from HEK293 cellstransfected with plasmids as indicated, was protected by both the pA1D2and pA3 probes. Lane M, 32P-labeled RNA markers (Qiu et al., 2002), withsizes indicated to the left. The origins of the protected bands in thelanes are indicated to the left and right for pA1D2 and pA3 probes,respectively. Spl, spliced RNAs; Unspl, unspliced RNAs. C)Quantification of the mRNAs spliced at the A3 splice acceptor. The bandsof both spliced and unspliced RNAs in lanes 4-6 of panel B werequantified. The ratio of spliced vs. unspliced RNA (A3 Spl/Unspl) wascalculated and is shown with means and standard deviations from threeindependent experiments. Numbers shown are P values calculated using atwo-tailed Student's t test. D) Northern blot analysis of cytoplasmic VPmRNAs. HEK293 cells were transfected with plasmids as indicated, andwere harvested at two days post-transfection. Cytoplasmic RNA wasextracted, and the RNA samples were analyzed by Northern blotting usingthe Cap probe. EB-stained 18S rRNA and VP mRNA are indicated. Asterisk(*) denotes various NS-encoding mRNAs. E) Western blot analysis ofcapsid proteins. Transfected cells were harvested and lysed at 2 dayspost-transfection. The lysates were analyzed by Western blotting usingan anti-VP antibody. The blot was reprobed with anti-β-actin. Thelysates were also analyzed by Western blotting using anti-NP1 andanti-HA antibodies. The identities of detected proteins are shown at theleft of the blot. F)-H) VP mRNA was exported from the nucleus tocytoplasm efficiently and was stable. F) Northern blot analysis. HEK293cells were transfected with plasmids as indicated with or withoutco-transfection of the pOpt-NP1. The same numbers of cells wereharvested and extracted for both total and cytoplasmic RNA. RNA sampleswere analyzed by Northern blotting using the Cap probe. EB-stained 18SrRNA and VP mRNA are indicated. Asterisk (*) denotes various NS617encoding mRNAs. G) Quantification of VP mRNAs on Northern blots. Thebands of VP mRNA in each lane of panel F were quantified and normalizedto the level of 18S rRNA. The intensity of the VP mRNA band in lane 1was arbitrally set as 100%. Relative VP mRNA level was calculated forthe bands in other lanes. Means and standard deviations are quantifiedfrom three independent experiments. P values shown are calculated usinga two-tailed Student's t-est. ND, not detectable. (H) RNA stabilityassay. HEK293 cells were transfected with pCMVNS*Cap. At 2 dayspost-infection, cells were treated with actinomycin D at 5 μg/mL forhours (hrs p.t.) as indicated. The treated cells were harvested andextracted for total RNA. The RNA samples were analyzed by Northernblotting using the NSCap probe (FIG. 1B). EB-stained 18S rRNA bands areshown. Indicated bands are detected VP mRNA at about 2.5 kb, which islikely the R6 mRNA that is polyadenylated at the (pA)d site, and (pA)pmRNA at about 1.5 kb, which is the R5s mRNA that is polyadenylated atthe (pA)p site (FIG. 1A). Asterisk (*) denotes various NS-encodingmRNAs. Control, total RNA of non-transfected cells.

FIGS. 7A-E. NP1 increased VP mRNA production independent of RNA splicingat the A3 splice acceptor. A) Diagrams of HBoV1 intron deletion/exchangeconstructs. Plasmids pCMVNS*(ln3Δ)Cap, pCMVEpoln14(In3A)Cap andpCMVEpoln124Cap are diagrammed with replaced introns shown. B) Westernblot analysis of capsid proteins. HEK293 cells were transfected withplasmids as indicated. Cells were harvested and lysed at 2 dayspost-transfection. The lysates were analyzed by Western blotting usingan anti-VP antibody and reprobed with anti-β-actin. The lysates werealso analyzed by Western blotting using anti-NP1 and anti-HA antibodies.C) Northern blot analysis of VP mRNAs. HEK293 cells were transfectedwith plasmids as indicated. Cells were harvested and extracted for totalRNA at 2 days post-transfection. The total RNA samples were analyzed byNorthern blotting using the NSCap probe. EB-stained 18S rRNA bands areshown. Detected bands of VP mRNA and (pA)p mRNA are indicated. Asterisk(*) denotes various NS-encoding mRNAs. Lane M, an RNA ladder marker. D)Quantification of VP mRNAs on Northern blots. The bands of VP mRNA and(pA)p mRNA in each lane of panel C were quantified and normalized to thelevel of 18S rRNA, respectively. The intensity of the VP mRNA band inlane 4 was arbitrally set as 100%. Relative intensities were calculatedfor the bands in other lanes. Means and standard deviations arecalculated from three independent experiments. P values shown arecalculated using a two-tailed Student's t test. Asterisks (**) denoteP<0.01. E) Determination of the usage of the A1, D2, and A3 splice sitesusing RPA. Ten μg of total RNA isolated at two days post-transfectionfrom HEK293 cells transfected with plasmids as indicated, was protectedby the pA1D2 and pA3 probes or their homology counterparts, asindicated. Lane M, 32P-labeled RNA markers (Qiu et al., 2002), withsizes indicated to the left. The origins of the protected bands areshown with sizes. Spl, spliced RNAs; Unspl, unspliced RNAs.

FIGS. 8A-D. NP1 increased VP mRNA production from cDNA constructs. A)Diagrams of HBoV1 cDNA constructs. HBoV1 cDNA constructs are diagrammedalong with the pCMVNS*(NP*)Cap control. B) Northern blot analysis of VPand (pA)p mRNAs. HEK293 cells were transfected with plasmids asindicated. Cells were harvested and extracted for total RNA at 2 dayspost-transfection. The RNA samples were analyzed by Northern blottingusing the NSCap probe. EB-stained 18S rRNA bands of each sample areshown. The identities of detected bands are indicated. Asterisk (*)denotes various NS-encoding mRNAs. C) Quantification of VP and (pA)pmRNAs on Northern blots. The bands of VP mRNA and (pA)p mRNA in eachlane of panel B were quantified and normalized to the level of 18S rRNA.The intensity of the VP mRNA band in lane 8 was arbitrally set as 100%.Relative intensity was calculated for the bands in other lanes. Meansand standard deviations are calculated from three independentexperiments. P values shown are calculated using a two-tailed Student'st test. Asterisks (**) indicate P<0.01. ND, not detectable. D) Westernblot analysis of capsid proteins. HEK293 cells were transfected withplasmids as indicated. Cells were harvested and lysed at 2 dayspost-transfection. The lysates were analyzed by Western blotting usingan anti-VP antibody. The blot was reprobed using an anti-β-actinantibody. The lysates were also analyzed by Western blotting using ananti-HA antibody.

FIGS. 9A-D. Mutation of the (pA)p sites enables VP mRNA production andcapsid protein expression in the absence of NP1. A) Diagrams of HBoV1NSCap and cDNA constructs. HBoV1 NS and Cap genes and cDNA constructsare diagrammed along with the pCMVNS*(NP*)Cap control. B) Northern blotanalysis of VP and (pA)p mRNAs. HEK293 cells were transfected withplasmids as indicated. The total RNA samples were analyzed by Northernblotting using the NSCap probe. EB-stained 18S rRNA bands are shown.Detected bands of VP and (pA)p mRNAs are indicated to the left of theblot. Asterisk (*) denotes various NS-encoding mRNAs. C) Quantificationof VP and (pA)p mRNAs on Northern blots. The bands of VP and (pA)p mRNAsin each lane in panel B were quantified and normalized to 18S rRNA. Theintensity of the VP mRNA band in lane 1 was arbitrally set as 100%.Relative intensity was calculated for the bands of both VP and (pA)pmRNAs in other lanes. Means and standard deviations are calculated fromthree independent experiments. P values shown are calculated using atwo-tailed Student's t test. Asterisks (*) indicate P<0.01. D) Westernblot analysis of capsid proteins. HEK293 cells were transfected withplasmids as indicated. Cell lysates of each transfection were analyzedby Western blotting using an anti-VP antibody. The blot was reprobedwith an anti-f-actin antibody. The lysates were also analyzed by Westernblotting using an anti-HA antibody for mCherry expression.

FIGS. 10A-C. Both purified wild type HBoV1 virions and purifiedrAAV2/HBoV1 vectors do not contain any nonstructural proteins (NS1, NS2,NS2, NS4 and NP1). 8×10⁹ DPR (DNAse I digestion resistant particles) ofwild type of HBoV1 (HBoV1) and rAAV2/HBoV1 vector rAAV2/HBoV1), whichwere purified by CsCl density-gradient centrifugation, were lyzed andseparated on SDS-10% PAGE. After transferring, the blots were probedusing anti-HBoV1 VP (Panel A) and anti-NS1 C-terminus (Panel B),respectively. The blot shown in panel B was reprobed with anti-HBoV1 NP1(Panel C). An amount of 0.5 million of 293 cells transfected withpHBV1NSCap plasmid (pHBoV1NSCap) or not transfected (Control) were lyzedand analyzed as positive and negative controls, respectively. Theidentity of each band detected is indicated to the right side of theblots. A lane with protein markers is shown with their respective sizesto the left side of the blot. Asterisk indicates non-specific bands.

FIG. 11. HBoV1 Genome Structure and Elements Used in rAAV/HBoV1 Helpers.LEH and REH represent the left and right end hairpin sequence at thetermini of HBoV1 genome (GeneBank Accession no: JQ923422, which isincorporated by reference herein). NS1, NS2, NS3, NS4, NP1 are thenon-structural protein: VP1, VP2 and VP3 are the capsid protein. All theHBoV1 encoded viral proteins are processed from a single preRNAtranscribed from P5 promoter. (pA)p and (pA)d indicated the proximal anddistal polyadenylation sites, respectively. NP1 plays a role for VPencoded mRNA splicing and read through the (pA)p sides. LEH and REHdeleted HBoV1 genome is used as the prototype packaging helper,pHBoVNScap for rAAV2/HBoV1 vector production. Sites to be mutated toin-frame stop codon for early termination of the non-structural proteinexpression are marked in red. Mutation at the D1′ splicing donorprevents NP1 expression. Codon optimization of the HBoV1 capsid proteinreading frame removes the five (pA)p sides in the unique region of the5′ end of VP1 and VP2. OptVP1 or OptVP1U indicate regions of codonoptimization of the full capsid coding sequence or only at the VP1/2 5′unique region. An optimized Kozak sequence (GTT AAG ACG) is used inoptVP1 and optVP1U to express VP1, and with the GUG codon for VP2remaining intact, in order to obtain an appropriate ratio of theVP1:VP2:VP3.

FIGS. 12A-D. Optimized Sequences. A) Optimized NP1 sequence (SEQ IDNO:1). B) Optimized VP sequence (SEQ ID NO:2). Translation starts at analternative start codon ACG of VP1, GTG for VP2, and ATG for VP3 (solidline box). VP1-3 are encoded in the same OFR; the short dashed line boxshows the unique region of VP1, the long dashed line box shows theunique region of VP2. Bold: VP2; bold and underlined: VP3. Upper Cases:codon optimized. C) Optimized VP sequence (SEQ ID NO:3). Translationstarts at an alternative start codon ACG of VP1, GTG for VP2, and ATGfor VP3 (solid line box). VP1-3 are encoded in the same OFR; the shortdashed line box shows the unique region of VP1, the long dashed line boxshows the unique region of VP2. Bold: VP2; bold and underlined: VP3.Upper Cases: codon optimized, lower cases: original coding sequence inthe HBoV1 genome. D) Exemplary wild-type VP sequence (SEQ ID NO:4).Translation starts at ATG for VP1, an alternative start codon GTG forVP2, and ATG for VP3 (solid line box). VP1-3 are encoded in the sameOFR; the short dashed line box shows the unique region of VP1, the longdashed line box shows the unique region of VP2. Bold: VP2; bold andunderlined: VP3.

DETAILED DESCRIPTION Definitions

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell,either in vitro or in vivo. Illustrative vectors include, for example,plasmids, viral vectors, liposomes and other gene delivery vehicles. Thepolynucleotide to be delivered, sometimes referred to as a “targetpolynucleotide” or “transgene,” may comprise a coding sequence ofinterest in gene therapy (such as a gene encoding a protein oftherapeutic or interest), a coding sequence of interest in vaccinedevelopment (such as a polynucleotide expressing a protein, polypeptideor peptide suitable for eliciting an immune response in a mammal),and/or a selectable or detectable marker.

“AAV” is adeno-associated virus, and may be used to refer to thenaturally occurring wild-type virus itself or derivatives thereof. Theterm covers all subtypes, serotypes and pseudotypes, and both naturallyoccurring and recombinant forms, except where required otherwise. Asused herein, the term “serotype” refers to an AAV which is identified byand distinguished from other AAVs based on capsid protein reactivitywith defined antisera, e.g., there are nine serotypes of primate AAVs,AAV-1 to AAV-9, although there are many related AAVs. For example,serotype AAV2 is used to refer to an AAV which contains capsid proteinsencoded from the cap gene of AAV 2 and a genome containing 5′ and 3′ ITRsequences from the same AAV2 serotype. For each example illustratedherein the description of the vector design and production describes theserotype of the capsid and 5′-3′ ITR sequences. The abbreviation “rAAV”refers to recombinant adeno-associated virus, also referred to as arecombinant AAV vector (or “rAAV vector”). Any AAV may be employed inthe production of chimeras, including both human and nonhuman primateAAVs.

BoV is bocavirus, and may be used to refer to the naturally occurringwild-type virus itself or derivatives thereof. The term covers allsubtypes, serotypes and pseudotypes, and both naturally occurring andrecombinant forms, except where required otherwise. As used herein, theterm “serotype” refers to a BoV, which is identified by anddistinguished from other BoVs based on capsid protein reactivity withdefined antisera, e.g., there are four known serotypes of humanbocavirus (HBoV), HBoV1, HBoV2, HBoV3, and HBoV4. However, included inBoV are serotypes derived from other non-human mammals such as swineBoV. Like for AAV, different serotypes of HBoV and BoV can havedifferent tropisms that infect different cell types and organs.

rAAV/HBoV is a chimeric vector which is composed of HBoV capsids and arAAV genome. In such a chimeric virus there is no genetic informationfrom HBoV within the genome. The rAAV genome may be from any serotype ofAAV.

rAAV/BoV is a chimeric vector which is composed of a non-human BoVcapsids and a rAAV genome. In such a chimeric virus there is no geneticinformation from BoV within the genome. The rAAV genome may be from anyserotype of AAV.

Tropism as used herein, is a term referring to the ability of aparticular viral serotype to productively infect cells of differingphenotypes or organs to deliver their genomic information to thenucleus.

“Transduction” or transducing as used herein, are terms referring to aprocess for the introduction of an exogenous polynucleotide, e.g., atransgene in rAAV vector, into a host cell leading to expression of thepolynucleotide, e.g., the transgene in the cell. The process includesone or more of 1) endocytosis of the chimeric virus, 2) escape fromendosomes or other intracellular compartments in the cytosol of a cell,3) trafficking of the viral particle or viral genome to the nucleus, 4)uncoating of the virus particles, and generation of expressible doublestranded AAV genome forms, including circular intermediates. The rAAVexpressible double stranded form may persist as a nuclear episome oroptionally may integrate into the host genome. The alteration of any ora combination of endocytosis of the chimeric virus after it has bound toa cell surface receptor, escape from endosomes or other intracellularcompartments to the cytosol of a cell, trafficking of the viral particleor viral genome to the nucleus, or uncoating of the virus particles, andgeneration of expressive double stranded AAV genome forms, includingcircular intermediates, by an agent of the invention, e.g., a proteasomeinhibitor, may result in altered expression levels or persistence ofexpression, or altered trafficking to the nucleus, or altered types orrelative numbers of host cells or a population of cells expressing theintroduced polynucleotide. Altered expression or persistence of apolynucleotide introduced via the chimeric virus can be determined bymethods well known to the art including, but not limited to, proteinexpression, e.g., by ELISA, flow cytometry and Western blot, measurementof and DNA and RNA production by hybridization assays, e.g., Northernblots, Southern blots and gel shift mobility assays. The agents of theinvention may alter, enhance or increase viral endocytosis, escape fromendosomes or other intracellular cytosolic compartments, and traffickinginto or to the nucleus, uncoating of the viral particles in the nucleus,and/or increasing concatamerization or generation of double strandedexpressible forms of the rAAV genome in the nucleus, so as to alterexpression of the introduced polynucleotide, e.g., a transgene in a rAAVvector, in vitro or in vivo. Methods used for the introduction of theexogenous polynucleotide include well-known techniques such astransfection, lipofection, viral infection, transformation, andelectroporation, as well as non-viral gene delivery techniques. Theintroduced polynucleotide may be stably or transiently maintained in thehost cell.

“Gene delivery” refers to the introduction of an exogenouspolynucleotide into a cell for gene transfer, and may encompasstargeting, binding, uptake, transport, localization, repliconintegration and expression.

“Gene transfer” refers to the introduction of an exogenouspolynucleotide into a cell which may encompass targeting, binding,uptake, transport, localization and replicon integration, but isdistinct from and does not imply subsequent expression of the gene.

“Gene expression” or “expression” refers to the process of genetranscription, translation, and post-translational modification.

A “detectable marker gene” is a gene that allows cells carrying the geneto be specifically detected (e.g., distinguished from cells which do notcarry the marker gene). A large variety of such marker genes are knownin the art.

A “selectable marker gene” is a gene that allows cells carrying the geneto be specifically selected for or against, in the presence of acorresponding selective agent. By way of illustration, an antibioticresistance gene can be used as a positive selectable marker gene thatallows a host cell to be positively selected for in the presence of thecorresponding antibiotic. A variety of positive and negative selectablemarkers are known in the art, some of which are described below.

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In preferred vector constructs of thisinvention, the heterologous polynucleotide is flanked by one or two AAVinverted terminal repeat sequences (ITRs). The term rAAV vectorencompasses both rAAV vector particles and rAAV vector plasmids.

A “Chimeric virus” or “Chimeric viral particle” refers to a viralparticle composed of at least one capsid protein and an encapsidatedpolynucleotide, which is from a different virus.

A “helper virus” for AAV refers to a virus that allows AAV (e.g.,wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpes viruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC.

An “infectious” virus or viral particle is one that comprises apolynucleotide component, which it is capable of delivering into a cellfor which the viral species is trophic. The term does not necessarilyimply any replication capacity of the virus.

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated or capped nucleotides and nucleotide analogs, and maybe interrupted by non-nucleotide components. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The term polynucleotide, as used herein, refersinterchangeably to double- and single-stranded molecules. Unlessotherwise specified or required, any embodiment of the inventiondescribed herein that is a polynucleotide encompasses both thedouble-stranded form and each of two complementary single-stranded formsknown or predicted to make up the double-stranded form.

A “transcriptional regulatory sequence” or “TRS,” as used herein, refersto a genomic region that controls the transcription of a gene or codingsequence to which it is operably linked. Transcriptional regulatorysequences of use in the present invention generally include at least onetranscriptional promoter and may also include one or more enhancersand/or terminators of transcription.

“Operably linked” refers to an arrangement of two or more components,wherein the components so described are in a relationship permittingthem to function in a coordinated manner. By way of illustration, atranscriptional regulatory sequence or a promoter is operably linked toa coding sequence if the TRS or promoter promotes transcription of thecoding sequence. An operably linked TRS is generally joined in cis withthe coding sequence, but it is not necessarily directly adjacent to it.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared. For example, apolynucleotide introduced by genetic engineering techniques into adifferent cell type is a heterologous polynucleotide (and, whenexpressed, can encode a heterologous polypeptide). Similarly, a TRS orpromoter that is removed from its native coding sequence and operablylinked to a different coding sequence is a heterologous TRS or promoter.

“Packaging” as used herein refers to a series of subcellular events thatresults in the assembly and encapsidation of a viral vector. Thus, whena suitable vector is introduced into a packaging cell line underappropriate conditions, it can be assembled into a viral particle.Functions associated with packaging of viral vectors are describedherein and in the art.

A “terminator” refers to a polynucleotide sequence that tends todiminish or prevent read-through transcription (i.e., it diminishes orprevent transcription originating on one side of the terminator fromcontinuing through to the other side of the terminator). The degree towhich transcription is disrupted is typically a function of the basesequence and/or the length of the terminator sequence. In particular, asis well known in numerous molecular biological systems, particular DNAsequences, generally referred to as “transcriptional terminationsequences,” are specific sequences that tend to disrupt read-throughtranscription by RNA polymerase, presumably by causing the RNApolymerase molecule to stop and/or disengage from the DNA beingtranscribed. Typical examples of such sequence-specific terminatorsinclude polyadenylation (“polyA”) sequences, e.g., SV40 polyA. Inaddition to or in place of such sequence-specific terminators,insertions of relatively long DNA sequences between a promoter and acoding region also tend to disrupt transcription of the coding region,generally in proportion to the length of the intervening sequence. Thiseffect presumably arises because there is always some tendency for anRNA polymerase molecule to become disengaged from the DNA beingtranscribed, and increasing the length of the sequence to be traversedbefore reaching the coding region would generally increase thelikelihood that disengagement would occur before transcription of thecoding region was completed or possibly even initiated. Terminators maythus prevent transcription from only one direction (“uni-directional”terminators) or from both directions (“bi-directional” terminators), andmay be comprised of sequence-specific termination sequences orsequence-non-specific terminators or both. A variety of such terminatorsequences are known in the art; and illustrative uses of such sequenceswithin the context of the present invention are provided below.

“Host cells,” “cell lines,” “cell cultures,” “packaging cell line” andother such terms denote higher eukaryotic cells, e.g., mammalian cells,such human cells, useful in the present invention. These cells can beused as recipients for recombinant vectors, viruses or other transferpolynucleotides, and include the progeny of the original cell that wastransduced. It is understood that the progeny of a single cell may notnecessarily be completely identical (in morphology or in genomiccomplement) to the original parent cell.

A “therapeutic gene,” “prophylactic gene,” “target polynucleotide,”“transgene,” “gene of interest” and the like generally refer to a geneor genes to be transferred using a vector. Typically, in the context ofthe present invention, such genes are located within the rAAV vector(which vector is flanked by inverted terminal repeat (ITR) regions andthus can be replicated and encapsidated into rAAV particles). Targetpolynucleotides can be used in this invention to generate rAAV vectorsfor a number of different applications. Such polynucleotides include,but are not limited to: (i) polynucleotides encoding proteins useful inother forms of gene therapy to relieve deficiencies caused by missing,defective or sub-optimal levels of a structural protein or enzyme; (ii)polynucleotides that are transcribed into anti-sense molecules; (iii)polynucleotides that are transcribed into decoys that bind transcriptionor translation factors; (iv) polynucleotides that encode cellularmodulators such as cytokines; (v) polynucleotides that can makerecipient cells susceptible to specific drugs, such as the herpes virusthymidine kinase gene; and (vi) polynucleotides for cancer therapy, suchas E1A tumor suppressor genes or p53 tumor suppressor genes for thetreatment of various cancers. To effect expression of the transgene in arecipient host cell, it is operably linked to a promoter, either its ownor a heterologous promoter. A large number of suitable promoters areknown in the art, the choice of which depends on the desired level ofexpression of the target polynucleotide; whether one wants constitutiveexpression, inducible expression, cell-specific or tissue-specificexpression, etc. The rAAV vector may also contain a selectable marker.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated.

“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction and/or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, including replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter. Promotersinclude AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as wellas heterologous (to parvovirus) promoters, e.g., a CMV promoter, abeta-actin promoter, a RSV-LTR or an ubiquitin promoter.

An “expression vector” is a vector comprising a region which encodes apolypeptide of interest, and is used for effecting the expression of theprotein in an intended target cell. An expression vector also comprisescontrol elements operatively linked to the encoding region to facilitateexpression of the protein in the target. The combination of controlelements and a gene or genes to which they are operably linked forexpression is sometimes referred to as an “expression cassette,” a largenumber of which are known and available in the art or can be readilyconstructed from components that are available in the art.

“Genetic alteration” refers to a process wherein a genetic element isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. The genetic element may be introduced into achromosome or mini-chromosome in the cell; but any alteration thatchanges the phenotype and/or genotype of the cell and its progeny isincluded in this term.

A cell is said to be “stably” altered, transduced or transformed with agenetic sequence if the sequence is available to perform its functionduring extended culture of the cell in vitro. In some examples, such acell is “inheritably” altered in that a genetic alteration is introducedwhich is also inheritable by progeny of the altered cell.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to polymers of amino acids of any length. The terms also encompassan amino acid polymer that has been modified; for example, disulfidebond formation, glycosylation, acetylation, phosphorylation, lipidation,or conjugation with a labeling component. Polypeptides such as “CFTR”and the like, when discussed in the context of gene therapy andcompositions therefor, refer to the respective intact polypeptide, orany fragment or genetically engineered derivative thereof, that retainsthe desired biochemical function of the intact protein. Similarly,references to CFTR, and other such genes for use in gene therapy(typically referred to as “transgenes” to be delivered to a recipientcell), include polynucleotides encoding the intact polypeptide or anyfragment or genetically engineered derivative possessing the desiredbiochemical function.

An “isolated” plasmid, virus, or other substance refers to a preparationof the substance devoid of at least some of the other components thatmay also be present where the substance or a similar substance naturallyoccurs or is initially prepared from. Thus, for example, an isolatedsubstance may be prepared by using a purification technique to enrich itfrom a source mixture. Enrichment can be measured on an absolute basis,such as weight per volume of solution, or it can be measured in relationto a second, potentially interfering substance present in the sourcemixture.

A preparation of AAV is said to be “substantially free” of helper virusif the ratio of infectious AAV particles to infectious helper virusparticles is at least about 10²:1; e.g., at least about 10⁴:1, includingat least about 10⁶:1 or at least about 10⁸:1. Preparations may also befree of equivalent amounts of helper virus proteins (i.e., proteins aswould be present as a result of such a level of helper virus if thehelper virus particle impurities noted above were present in disruptedform). Viral and/or cellular protein contamination can generally beobserved as the presence of Coomassie blue staining bands on SDS gels(e.g., the appearance of bands other than those corresponding to the AAVcapsid proteins VP1, VP2 and VP3).

“Efficiency” when used in describing viral production, replication orpackaging refers to useful properties of the method: in particular, thegrowth rate and the number of virus particles produced per cell. “Highefficiency” production indicates production of at least 100 viralparticles per cell; e.g., at least about 10,000 or at least about100,000 particles per cell, over the course of the culture periodspecified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, virology,microbiology, recombinant DNA, and immunology, which are within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Sambrook et al., 1989; Gait, 1984; Freshney, 1987; the seriesMethods in Enzymology (Academic Press, Inc.); Miller et al., 1987; Weiret al., 1996; Ausubel et al., 1998; Coligan et al., 1991; Coligan etal., 1995; and Scopes 1994.

I. Chimeric Viruses

Human airway epithelial cells are highly resistant to infection by mostviral vectors included the adeno-associated virus (rAAV), the mostwidely used gene therapy vector in clinical trials. Human Bocavirus 1(HBoV1), an autonomous human parvovirus which is likely an etiologicalagent of acute respiratory tract infections (ARTI) associated withwheezing in infants and young children (Allander et al., 2007;Christensen et al., 2010; Deng et al., 2012; Don et al., 2010),efficiently infects HAE from the apical membrane, resulting inreplication of progeny viruses and cytopathology (Huang et al., 2012a).Impressively, HBoV1 infection of HAE at extremely low multiplicities ofinfection (MOI) of 10⁻³ DNAse-resistant particles (DRP) per cell resultsin a productive infection (see Example 2). Recently, the full-length5543-nt HBoV1 complete genome (including terminal palindromic sequencesat both ends) was cloned, and cell culture systems for HBoV1 productionhave been established (Example 1). Given the high efficiency of HBoV1infection from the apical surface of HAE, HoBV1 was hypothesized to besuitable for engineering recombinant vectors for human airway genetherapy.

HBoV1 is a relative of AAV and other Parvoviridae family members. HBoV1belongs to the genus Bocavirus, while AAV is in the genus Dependovirus(Tijssen et al., 2011). HBoV1 and AAV are both small single-stranded DNAviruses, but 90% of encapsidated HBoV1 genomes are of the minus strand,while for AAV, an equal ratio of plus and minus strands are encapsidated(Schildgen et al., 2012). These two viruses differ greatly in theirlytic phase life cycle; AAV requires co-infection with a helper virus,while HBoV1 autonomously replicates progeny in permissive cells (Huanget al., 2012a; Dijkman et al., 2009). The HBoV1 genome size is 5543 nt,18.5% (863 nt) larger than that of AAV2 (4679-nt), and its structuralfeatures include asymmetrical hairpins with unique palindromic sequencesat 5′ (140 nt) and 3′ (200 nt) termini, which are involved inreplication and encapsidation, and a single P5 promoter that transcribesall viral structural and non-structural proteins (Huang et al., 2012;Chen et al., 2010). This is in contrast to the inverted terminal repeatsand multiple internal promoters found in AAV genomes. HBoV1 capsidsurface topology possesses common features with other parvoviruses(icosahedral capsid), and is most closely similar to human parvovirusB19 (Gurda et al., 2010). Uke the cloned AAV genome, a plasmid thatencodes the HBoV1 proviral genome is infectious and can be used toproduce infectious particles through transfection into HEK 293 cellswithout the need for helper virus co-infection.

Cross-genera pseudopackaging between Parvoviridae was first establishedwhen a rAAV genome was encapsidated into a human parvovirus B19 capsid(Ponnazhagan et al., 1998). This resultant cross-genera chimera was ableto deliver the rAAV genome into human bone marrow cells that areresistant to rAAV infection (Ponnazhagan et al., 1998). One importantfeature of the HBoV1 virus is that its genome autonomously replicates inpermissive cells, in contrast to rAAV, which is a dependent parvovirusand needs helper virus coinfection for replication.

With the success in trans-complementation for rHBoV1 vector production,a so-called replicative rHBoV1 vector was developed by retaining thecoding sequences for HBoV1 rep genes but replacing the structural geneby a transgene. This type of vector can deliver a high level oftherapeutic gene expression in the airway cells for the therapy such asCF, AAT deficiency, COPD, or lung cancers. Such a replicating HBoV1vector could have high utility as a vaccine against WT HBoV1 infections.

Another vector developed was an AAV2/HBoV1 chimeric virus, whichpackages a rAAV2 genome into a HBoV1 capsid particle. The vector wasalso produced in HEK293 cells with a procedure similar for rAAV vector,but the capsid genes are substituted by HBoV1 capsids. This AAV/HBoV1vector combines both the advantages of AAV and HBoV1 transductionbiology, with less safety concerns than the rHBoV1 vector since rAAVvector genomes have been extensively studied in many pre-clinicalresearch and clinical trials, but higher airway cell tropism than rAAV.More importantly, the large HBoV1 package capacity makes it possible toencapsidate an oversized rAAV genome up to about 5.5 kb or about 6.0 kb.The 20% greater capacity than rAAV is enough to house a strongexpression cassette for effective gene expression. A rAAV genomeprovides advantages of persistent gene expression by the stable circulartransduction intermediates and double stranded genome concatemers.Indeed, AAV/HBoV1 vectors featured more persistent transgene expressionthan the rHBoV1 vector. Furthermore, the rescue and replication of rAAVgenomes in HEK293 cells was very efficient, so that the production yieldof the AAV/HBoV1 vector was also better than an rHBoV1 vector.

Utilizing the larger packaging capacity of HBoV1, a rAAV2/HBoV1-CFTRvector was previously prepared that harbors a 5.5 kb oversized rAAVgenome with a 5.2 kb CFTR expression cassette having a strong chimericpromoter that included the human CMV immediate gene enhancer and thechicken β-actin promoter (CBA promoter). That vector demonstrated about30% restoration of CFTR-mediate chloride currents in CF HAE followingapical infection. Therefore, the vector could efficiently deliver normalCFTR protein expression on the surface of the airway epithelial cellsand correct the defective CFTR specific chloride transport in the CFHAE. In addition, the HBoV1 genome can encapsidate theself-complementary double stranded form of a rAAV genome of about 2.7 kbto about 2.8 kb in length, which vector can bypass genome conversion andallow for enhanced or more rapid transgene expression. The AAV/HBoVchimeric vectors could also be expanded to other therapies for otherlung diseases such as alpha-antitrypsin deficiency, asthma, and lungcancer, as well as vaccination against wild-type HBoV infections ininfants. However, the yields of chimeric virus produced were notsuitable for gene therapy applications, in contrast to the vectors andmethods described herein.

The capsids and/or genomes of the viruses of the invention may bechimeric, e.g., as a result of directed evolution (see, e.g., U et al.,2009).

II. rAAV Vectors

Besides prophylactic or therapeutic gene products, recombinant AAVvectors and/or viruses can also comprise polynucleotides that do notencode proteins, including, e.g., polynucleotides encoding for antisensemRNA (the complement of mRNA) which can be used to block the translationof normal mRNA by forming a duplex with it, and polynucleotides thatencode ribozymes (RNA catalysts). In addition selected pairs of rAAVvectors having portions of open reading frames flanked by appropriatelyplaced splice acceptor sites and/or splice donor sites, or havingtranscription regulatory sequences such as a heterologous enhancer, aheterologous promoter, or a heterologous enhancer and a promoter, may beemployed. See, e.g., U.S. Pat. No. 6,436,392, the disclosure of which isincorporated by reference herein. For example, a first AAV vector mayinclude a first DNA segment comprising a 5′-inverted terminal repeat ofAAV; a second DNA segment comprising a promoter operably linked to a DNAfragment comprising an exon of a gene and a splice donor site, whereinthe second DNA segment does not encode a full-length polypeptide; and athird DNA segment comprising a 3′-inverted terminal repeat of AAV; and asecond AAV vector comprising linked: a first DNA segment comprising a5′-inverted terminal repeat of AAV; a second DNA segment comprising asplice acceptor site and a DNA fragment with at least one other exonwhich together with the DNA segment of the first AAV vector encodes afull-length polypeptide; and a third DNA segment comprising a3′-inverted terminal repeat of AAV. In one example, a first AAV vectorincludes the following: a first nucleic acid segment comprising a5′-inverted terminal repeat of AAV; a second nucleic acid segmentcomprising a portion of a gene which includes a transcriptionalregulatory region; a third nucleic acid segment comprising a splicedonor site; and a fourth nucleic acid segment comprising a 3′-invertedterminal repeat of AAV; and a second AAV vector comprising linked: afirst nucleic acid segment comprising a 5′-inverted terminal repeat ofAAV; a second nucleic acid segment comprising a splice acceptor site; athird nucleic acid segment comprising a portion of a gene which togetherwith the nucleic acid segment of the first AAV vector comprises a genecomprising an open reading frame which encodes a functional polypeptide;and a fourth nucleic acid segment comprising a 3′-inverted terminalrepeat of AAV. In a further example, a first AAV vector includes thefollowing: a first nucleic acid segment comprising a 5′-invertedterminal repeat of AAV; a second nucleic acid segment comprising asplice acceptor site; a third nucleic acid segment comprising a portionof a gene; and a fourth nucleic acid segment comprising a 3′-invertedterminal repeat of AAV; and a second composition comprising a second AAVvector comprising: a first nucleic acid segment comprising a 5′-invertedterminal repeat of AAV; a second nucleic acid segment comprising aportion of a gene which together with the nucleic acid segment abovehaving the portion comprises a gene comprising an open reading framewhich encodes a functional polypeptide, wherein the portion of the geneincludes a transcriptional regulatory region; a third nucleic acidsegment comprising a splice donor site; a fourth nucleic acid segmentcomprising a 3′-inverted terminal repeat of AAV; which vectors in a hostcell yield a RNA transcript which comprises sequences from the first AAVvector linked to sequences from the second AAV vector, which sequencesare positioned so that the splice donor site is 5′ to the spliceacceptor site, and which transcript is spliced to a mRNA which encodesthe functional protein.

Adeno-associated viruses of any serotype are suitable to prepare rAAV,since the various serotypes are functionally and structurally related,even at the genetic level (see, e.g., Blacklow, 1988; and Rose, 1974).All AAV serotypes apparently exhibit similar replication propertiesmediated by homologous rep genes; and all generally bear three relatedcapsid proteins such as those expressed in AAV2. The degree ofrelatedness is further suggested by heteroduplex analysis which revealsextensive cross-hybridization between serotypes along the length of thegenome; and the presence of analogous self-annealing segments at thetermini that correspond to ITRs. The similar infectivity patterns alsosuggest that the replication functions in each serotype are undersimilar regulatory control. Among the various AAV serotypes, AAV2 ismost commonly employed.

An AAV vector of the invention typically comprises a polynucleotide thatis heterologous to AAV. The polynucleotide is typically of interestbecause of a capacity to provide a function to a target cell in thecontext of gene therapy, such as up- or down-regulation of theexpression of a certain phenotype. Such a heterologous polynucleotide or“transgene,” generally is of sufficient length to provide the desiredfunction or encoding sequence.

Where transcription of the heterologous polynucleotide is desired in theintended target cell, it can be operably linked to its own or to aheterologous promoter, depending for example on the desired level and/orspecificity of transcription within the target cell, as is known in theart. Various types of promoters and enhancers are suitable for use inthis context. Constitutive promoters provide an ongoing level of genetranscription, and may be preferred when it is desired that thetherapeutic or prophylactic polynucleotide be expressed on an ongoingbasis. Inducible promoters generally exhibit low activity in the absenceof the inducer, and are up-regulated in the presence of the inducer.They may be preferred when expression is desired only at certain timesor at certain locations, or when it is desirable to titrate the level ofexpression using an inducing agent. Promoters and enhancers may also betissue-specific: that is, they exhibit their activity only in certaincell types, presumably due to gene regulatory elements found uniquely inthose cells.

Illustrative examples of promoters are the SV40 late promoter fromsimian virus 40, the Baculovirus polyhedron enhancer/promoter element,Herpes Simplex Virus thymidine kinase (HSV tk), the immediate earlypromoter from cytomegalovirus (CMV) and various retroviral promotersincluding LTR elements. Inducible promoters include heavy metal ioninducible promoters (such as the mouse mammary tumor virus (mMTV)promoter or various growth hormone promoters), and the promoters from T7phage which are active in the presence of T7 RNA polymerase. By way ofillustration, examples of tissue-specific promoters include varioussurfactin promoters (for expression in the lung), myosin promoters (forexpression in muscle), and albumin promoters (for expression in theliver). A large variety of other promoters are known and generallyavailable in the art, and the sequences of many such promoters areavailable in sequence databases such as the GenBank database.

Where translation is also desired in the intended target cell, theheterologous polynucleotide will preferably also comprise controlelements that facilitate translation (such as a ribosome binding site or“RBS” and a polyadenylation signal). Accordingly, the heterologouspolynucleotide generally comprises at least one coding regionoperatively linked to a suitable promoter, and may also comprise, forexample, an operatively linked enhancer, ribosome binding site andpoly-A signal. The heterologous polynucleotide may comprise one encodingregion, or more than one encoding regions under the control of the sameor different promoters. The entire unit, containing a combination ofcontrol elements and encoding region, is often referred to as anexpression cassette.

The heterologous polynucleotide is integrated by recombinant techniquesinto or in place of the AAV genomic coding region (i.e., in place of theAAV rep and cap genes), but is generally flanked on either side by AAVinverted terminal repeat (ITR) regions. This means that an ITR appearsboth upstream and downstream from the coding sequence, either in directjuxtaposition, e.g., (although not necessarily) without any interveningsequence of AAV origin in order to reduce the likelihood ofrecombination that might regenerate a replication-competent AAV genome.However, a single ITR may be sufficient to carry out the functionsnormally associated with configurations comprising two ITRs (see, forexample, WO 94/13788), and vector constructs with only one ITR can thusbe employed in conjunction with the packaging and production methods ofthe present invention.

The native promoters for rep are self-regulating, and can limit theamount of AAV particles produced. The rep gene can also be operablylinked to a heterologous promoter, whether rep is provided as part ofthe vector construct, or separately. Any heterologous promoter that isnot strongly down-regulated by rep gene expression is suitable; butinducible promoters may be preferred because constitutive expression ofthe rep gene can have a negative impact on the host cell. A largevariety of inducible promoters are known in the art; including, by wayof illustration, heavy metal ion inducible promoters (such asmetallothionein promoters); steroid hormone inducible promoters (such asthe MMTV promoter or growth hormone promoters); and promoters such asthose from T7 phage which are active in the presence of T7 RNApolymerase. One sub-class of inducible promoters are those that areinduced by the helper virus that is used to complement the replicationand packaging of the rAAV vector. A number of helper-virus-induciblepromoters have also been described, including the adenovirus early genepromoter which is inducible by adenovirus E1A protein; the adenovirusmajor late promoter; the herpesvirus promoter which is inducible byherpesvirus proteins such as VP16 or 1CP4; as well as vaccinia orpoxvirus inducible promoters.

Methods for identifying and testing helper-virus-inducible promotershave been described (see, e.g., WO 96/17947). Thus, methods are known inthe art to determine whether or not candidate promoters arehelper-virus-inducible, and whether or not they will be useful in thegeneration of high efficiency packaging cells. Briefly, one such methodinvolves replacing the p5 promoter of the AAV rep gene with the putativehelper-virus-inducible promoter (either known in the art or identifiedusing well-known techniques such as linkage to promoter-less “reporter”genes). The AAV rep-cap genes (with p5 replaced), e.g., linked to apositive selectable marker such as an antibiotic resistance gene, arethen stably integrated into a suitable host cell (such as the HeLa orA549 cells exemplified below). Cells that are able to grow relativelywell under selection conditions (e.g., in the presence of theantibiotic) are then tested for their ability to express the rep and capgenes upon addition of a helper virus. As an initial test for rep and/orcap expression, cells can be readily screened using immunofluorescenceto detect Rep and/or Cap proteins. Confirmation of packagingcapabilities and efficiencies can then be determined by functional testsfor replication and packaging of incoming rAAV vectors. Using thismethodology, a helper-virus-inducible promoter derived from the mousemetallothionein gene has been identified as a suitable replacement forthe p5 promoter, and used for producing high titers of rAAV particles(as described in WO 96/17947).

Removal of one or more AAV genes is in any case desirable, to reduce thelikelihood of generating replication-competent AAV (“RCA”). Accordingly,encoding or promoter sequences for rep, cap, or both, may be removed,since the functions provided by these genes can be provided in trans.

The resultant vector is referred to as being “defective” in thesefunctions. In order to replicate and package the vector, the missingfunctions are complemented with a packaging gene, or a pluralitythereof, which together encode the necessary functions for the variousmissing rep and/or cap gene products. The packaging genes or genecassettes are in one embodiment not flanked by AAV ITRs and in oneembodiment do not share any substantial homology with the rAAV genome.Thus, in order to minimize homologous recombination during replicationbetween the vector sequence and separately provided packaging genes, itis desirable to avoid overlap of the two polynucleotide sequences. Thelevel of homology and corresponding frequency of recombination increasewith increasing length of homologous sequences and with their level ofshared identity. The level of homology that will pose a concern in agiven system can be determined theoretically and confirmedexperimentally, as is known in the art. Typically, however,recombination can be substantially reduced or eliminated if theoverlapping sequence is less than about a 25 nucleotide sequence if itis at least 80% identical over its entire length, or less than about a50 nucleotide sequence if it is at least 70% identical over its entirelength. Of course, even lower levels of homology are preferable sincethey will further reduce the likelihood of recombination. It appearsthat, even without any overlapping homology, there is some residualfrequency of generating RCA. Even further reductions in the frequency ofgenerating RCA (e.g., by nonhomologous recombination) can be obtained by“splitting” the replication and encapsidation functions of AAV, asdescribed by Allen et al., WO 98/27204).

The rAAV vector construct, and the complementary packaging geneconstructs can be implemented in this invention in a number of differentforms. Viral particles, plasmids, and stably transformed host cells canall be used to introduce such constructs into the packaging cell, eithertransiently or stably.

In certain embodiments of this invention, the AAV vector andcomplementary packaging gene(s), if any, are provided in the form ofbacterial plasmids, AAV particles, or any combination thereof. In otherembodiments, either the AAV vector sequence, the packaging gene(s), orboth, are provided in the form of genetically altered (preferablyinheritably altered) eukaryotic cells. The development of host cellsinheritably altered to express the AAV vector sequence, AAV packaginggenes, or both, provides an established source of the material that isexpressed at a reliable level.

A variety of different genetically altered cells can thus be used in thecontext of this invention. By way of illustration, a mammalian host cellmay be used with at least one intact copy of a stably integrated rAAVvector. An AAV packaging plasmid comprising at least an AAV rep geneoperably linked to a promoter can be used to supply replicationfunctions (as described in U.S. Pat. No. 5,658,776).

Alternatively, a stable mammalian cell line with an AAV rep geneoperably linked to a promoter can be used to supply replicationfunctions (see, e.g., Trempe et al., WO 95/13392); Burstein et al. (WO98/23018); and Johnson et al. (U.S. Pat. No. 5,656,785). The AAV capgene, providing the encapsidation proteins as described above, can beprovided together with an AAV rep gene or separately (see, e.g., theabove-referenced applications and patents as well as Allen et al. (WO98/27204). Other combinations are possible and included within the scopeof this invention.

III. Uses of Chimeric Virus

The chimeric virus produced by the methods described herein can be usedfor administration to an individual for purposes of gene therapy orvaccination. Suitable diseases for therapy include but are not limitedto those induced by viral, bacterial, or parasitic infections, variousmalignancies and hyperproliferative conditions, autoimmune conditions,and congenital deficiencies.

Gene therapy can be conducted to enhance the level of expression of aparticular protein either within or secreted by the cell. Vectors ofthis invention may be used to genetically alter cells either for genemarking, replacement of a missing or defective gene, or insertion of atherapeutic gene. Alternatively, a polynucleotide may be provided to thecell that decreases the level of expression. This may be used for thesuppression of an undesirable phenotype, such as the product of a geneamplified or overexpressed during the course of a malignancy, or a geneintroduced or overexpressed during the course of a microbial infection.Expression levels may be decreased by supplying a therapeutic orprophylactic polynucleotide comprising a sequence capable, for example,of forming a stable hybrid with either the target gene or RNA transcript(antisense therapy), capable of acting as a ribozyme to cleave therelevant mRNA or capable of acting as a decoy for a product of thetarget gene.

Vaccination can be conducted to protect cells from infection byinfectious pathogens. As the traditional vaccine methods, vectors ofthis invention may be used to deliver transgenes encoding viral,bacterial, tumor or fungal antigen and their subsequent expression inhost cells. The antigens, which expose to the immune system to evoke animmune response, can be in the form of virus-like particle vaccines orsubunit vaccines of virus-coding proteins. Alternatively, as the methodof passive immunization, vectors of this invention might be used todeliver genes encoding neutralizing antibodies and their subsequentexpression in host non-hematopoietic tissues. The vaccine-likeprotection against pathogen infection can be conducted through directprovision of neutralizing antibody from vector-mediated transgeneexpression, bypassing the reliance on the natural immune system formounting desired humoral immune responses.

The introduction of the chimeric vectors by the methods of the presentinvention may involve use of any number of delivery techniques (bothsurgical and non-surgical) which are available and well known in theart. Such delivery techniques, for example, include vascularcatheterization, cannulization, injection, inhalation, endotracheal,subcutaneous, inunction, topical, oral, percutaneous, intra-arterial,intravenous, and/or intraperitoneal administrations. Vectors can also beintroduced by way of bioprostheses, including, by way of illustration,vascular grafts (PTFE and dacron), heart valves, intravascular stents,intravascular paving as well as other non-vascular prostheses. Generaltechniques regarding delivery, frequency, composition and dosage rangesof vector solutions are within the skill of the art.

In particular, for delivery of a vector of the invention to a tissue,any physical or biological method that will introduce the vector to ahost animal can be employed. Vector means both a bare recombinant vectorand vector DNA packaged into viral coat proteins, as is well known foradministration. Simply dissolving a chimeric or rHBoV vector inphosphate buffered saline has been demonstrated to be sufficient toprovide a vehicle useful for muscle tissue expression, and there are noknown restrictions on the carriers or other components that can becoadministered with the vector (although compositions that degrade DNAshould be avoided in the normal manner with vectors). Pharmaceuticalcompositions can be prepared as injectable formulations or as topicalformulations to be delivered to the muscles by transdermal transport.Numerous formulations for both intramuscular injection and transdermaltransport have been previously developed and can be used in the practiceof the invention. The vectors can be used with any pharmaceuticallyacceptable carrier for ease of administration and handling.

For purposes of intramuscular injection, solutions in an adjuvant suchas sesame or peanut oil or in aqueous propylene glycol can be employed,as well as sterile aqueous solutions. Such aqueous solutions can bebuffered, if desired, and the liquid diluent first rendered isotonicwith saline or glucose. Solutions of the chimeric or rHBoV vector as afree acid (DNA contains acidic phosphate groups) or a pharmacologicallyacceptable salt can be prepared in water suitably mixed with asurfactant such as hydroxypropylcellulose. A dispersion of viralparticles can also be prepared in glycerol, liquid polyethylene glycolsand mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms. In this connection, the sterile aqueous mediaemployed are all readily obtainable by standard techniques well-known tothose skilled in the art.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of a dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the chimericor rHBoV vector in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the methods ofpreparation include but are not limited to vacuum drying and the freezedrying technique which yield a powder of the active ingredient plus anyadditional desired ingredient from the previously sterile-filteredsolution thereof.

For purposes of topical administration, dilute sterile, aqueoussolutions (usually in about 0.1% to 5% concentration), otherwise similarto the above parenteral solutions, are prepared in containers suitablefor incorporation into a transdermal patch, and can include knowncarriers, such as pharmaceutical grade dimethylsulfoxide (DMSO).

Of interest is the correction of the genetic defect of cystic fibrosis,by supplying a properly functioning cystic fibrosis transmembraneconductance regulator (CFTR) to the airway epithelium. Thus, the use ofchimeric or rHBoV vectors encoding native CFTR protein, and mutants andfragments thereof, is envisioned.

Compositions of this invention may be used in vivo as well as ex vivo.In vivo gene therapy comprises administering the vectors of thisinvention directly to a subject. Pharmaceutical compositions can besupplied as liquid solutions or suspensions, as emulsions, or as solidforms suitable for dissolution or suspension in liquid prior to use. Foradministration into the respiratory tract, one mode of administration isby aerosol, using a composition that provides either a solid or liquidaerosol when used with an appropriate aerosolubilizer device. Anothermode of administration into the respiratory tract is using a flexiblefiberoptic bronchoscope to instill the vectors. Typically, the viralvectors are in a pharmaceutically suitable pyrogen-free buffer such asRinger's balanced salt solution (pH 7.4). Although not required,pharmaceutical compositions may optionally be supplied in unit dosageform suitable for administration of a precise amount.

An effective amount of virus is administered, depending on theobjectives of treatment. An effective amount may be given in single ordivided doses. Where a low percentage of transduction can cure a geneticdeficiency, then the objective of treatment is generally to meet orexceed this level of transduction. In some instances, this level oftransduction can be achieved by transduction of only about 1 to 5% ofthe target cells, but is more typically 20% of the cells of the desiredtissue type, usually at least about 50%, at least about 80%, at leastabout 95%, or at least about 99% of the cells of the desired tissuetype. As a guide, the number of vector particles present in a singledose given by bronchoscopy will generally be at least about 1×10¹²,e.g., about 1×10¹³, 1×10¹⁴, 1×10¹⁵ or 1×10¹⁶ particles, including bothDNAse-resistant and DNAse-susceptible particles. In terms ofDNAse-resistant particles (DRPs), the dose will generally be between1×10¹² and 1×10¹⁶ particles, more generally between about 1×10¹² and1×10¹⁵ particles. The treatment can be repeated as often as every two orthree weeks, as required, although treatment once in 180 days may besufficient.

To confirm the presence of the desired DNA sequence in the host cell, avariety of assays may be performed. Such assays include, for example,“molecular biological” assays well known to those of skill in the art,such as Southern and Northern blotting, RT-PCR and PCR; “biochemical”assays, such as detecting the presence of a polypeptide expressed from agene present in the vector, e.g., by immunological means(immunoprecipitations, immunoaffinity columns, ELISAs and Western blots)or by any other assay useful to identify the presence and/or expressionof a particular nucleic acid molecule falling within the scope of theinvention.

To detect and quantitate RNA produced from introduced DNA segments,RT-PCR may be employed. In this application of PCR, it is firstnecessary to reverse transcribe RNA into DNA, using enzymes such asreverse transcriptase, and then through the use of conventional PCRtechniques amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique demonstrates the presence of an RNAspecies and gives information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and only demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect the DNA segment inquestion, they do not provide information as to whether the DNA segmentis being expressed. Expression may be evaluated by specificallyidentifying the polypeptide products of the introduced DNA sequences orevaluating the phenotypic changes brought about by the expression of theintroduced DNA segment in the host cell.

Thus, the effectiveness of the genetic alteration can be monitored byseveral criteria, including analysis of physiological fluid samples,e.g., urine, plasma, serum, blood, cerebrospinal fluid or nasal or lungwashes. Samples removed by biopsy or surgical excision may be analyzedby in situ hybridization, PCR amplification using vector-specificprobes, RNAse protection, immunohistology, or immunofluorescent cellcounting. When the vector is administered by bronchoscopy, lung functiontests may be performed, and bronchial lavage may be assessed for thepresence of inflammatory cytokines. The treated subject may also bemonitored for clinical features, and to determine whether the cellsexpress the function intended to be conveyed by the therapeutic orprophylactic polynucleotide.

The decision of whether to use in vivo or ex vivo therapy, and theselection of a particular composition, dose, and route of administrationwill depend on a number of different factors, including but not limitedto features of the condition and the subject being treated. Theassessment of such features and the design of an appropriate therapeuticor prophylactic regimen is ultimately the responsibility of theprescribing physician.

The foregoing description provides, inter alia, methods for generatinghigh titer preparations of recombinant chimeric viruses or rHBoV thatare substantially free of helper virus (e.g., adenovirus) and cellularproteins. It is understood that variations may be applied to thesemethods by those of skill in this art without departing from the spiritof this invention.

IV. Uses and Comparison of Vectors and Methods for AAV/BoV ChimeraProduction

CF is caused by single gene defect in the cystic fibrosis transmembraneconductance regulator (CFTR), is the most common life-threateninginherent disease with about $450 million spent annually on patient carein the U.S. alone. Gene therapy appears the best cure to permanentlytreat this disorder, regardless of the gene mutation/genotype, byreplacing the defective CFTR gene with a corrected gene. rAAV/BoV vectoralso can be applied for the gene therapy to treat other airway diseasescaused by inherited gene defects such as alpha-antitrypsin (AAT)deficiency and other chronic acquired respiratory disorders such asasthma. Other applications include the treatment lung cancers andprevention of infectious diseases caused by respiratory viruses throughvaccination.

Previously, a gene transfer vector, rAAV/HBoV1 for human pulmonarydisease gene therapy and vaccines, was constructed (Yan et al., 2013).rAAV/HBoV1 vector is highly efficient at transduction of the humanairway and has larger packaging capacity than rAAV vectors that use AAVcapsid. This chimeric vector solved the problem of the lack of efficientviral vector to transduce human airway, and is of great interest in thegene therapy application for cystic fibrosis (CF) due to its largerpackaging capacity. The system used a non-replicating HBoV nonstructural(NS) and capsid proteins (Cap)-expressing plasmid, pHBoV1NSCap, as thepackaging helper to produce rAAV/HBoV1 vector. The production yield ofpurified vector is 2×10¹¹ DRP (DNAse digestion-resistant particles) per40 (145 mm)-plates transfected HEK 293 cells, that is about 400 to 600DRP/cell (in crude lysate) or 250 DRP/cell (purified vector), and isabout 5 to 10% of the yield of rAAV2 vector production in transfected293 cells. This production yield is sufficient for routine studies inthe laboratory on vector biology and functional assays of transduction.For example, this system an AAV/HBoV vector was produced harboring a 5.5kb oversized rAAV genome with a 5.2 kb strong CFTR expression cassette.The function and efficacy of this novel CFTR vector has been validatedin cystic fibrosis (CF) polarized human airway epithelial cultures grownat an air-liquid interface (HAE-ALI). Following apical infection, thevector can efficiently deliver normal CFTR protein expression on thesurface of the airway epithelial cells and correct defective CFTRchloride transport defects in CF HAE-ALI cultures (Yan et al, 2013).However, for preclinical studies in experimental animals and futureclinical trial, this production yield is not sufficient.

Thus, prior to this disclosure, where an HBoV nonstructural and capsidproteins-expressing plasmid pHBoV1NSCap was used as helper tocross-genus pseudopackage rAAV genome in HBoV capsid, virus productionwas not sufficient enough for clinical application. The presentdisclosure reveals details of HBoV viral protein expression profiles andthe involvement of the HBoV non-structural proteins in capsid expressionand vector production. These findings are likely relevant to all HBoVserotypes from human as well as other mammalian bocoviruses (not justhuman HBoV1). Findings on the biological properties of HBoV that arerelevant to vector development include: 1) HBoV non-structural proteinNP1 plays a role in regulating HBoV capsid expression and rAAV/HBoVvector packaging; 2) knockout of the proximal polyadenylation sites((pA)p) in Cap coding sequence compensates the NP1 protein for capsidprotein expression; and 3) expression of non-structural proteins NS1,NS2, NS3 and NS4 is not essential for vector production. This disclosurealso relates to a series of new packaging helpers plasmids thatinactivate NS1-4 expression, the exchange of endogenous P5 promoter withthe CMV IE enhancer/promoter, as well as codon optimization of thecapsid protein-encoding sequence. A 5- to 75-fold increment in rAAV/HBoVproduction can be achieved using these helpers in HEK293 cell productionsystem, compared to the previous helper pHBoV1NSCap. These capsidexpression constructs can also be employed to the baculovirus/insectcells production system for rAAV/HBoV vector production.

The HBoV1 genetic map, which revealed a detail transcriptional profileof the HBoV1 NS and cap genes and the viral proteins expressed fromdifferent spliced mRNAs from the pre-RNA transcribed by the native P5promoter. (See FIGS. 1 and 11). While investigating the involvement ofthe HBoV non-structural protein on capsid protein expression, it wasfound that: 1) ectopic expression of the HBoV1 cap ORF is not sufficientto express capsid proteins, due to the poor production of mRNA in thecytoplasm; 2) non-structural protein NP1 is the critical componentrequired for regulating HBoV capsid expression, which facilitates thesplicing of capsid protein-encoding mRNA and thereby activates capsidexpression; 3) the expression of non-structural proteins NS1, NS2, NS3and NS4 are not essential for capsid protein expression; 4) internalpolyadenylation prevents HBoV1 pre-mRNA transcription through proximalpolyadenylation (pA)p sites within the capsid protein coding sequence,knockout of the (pA)p sites compensates for the requirement of NP1protein in the expression of capsid proteins.

The utility of these findings on HBoV1 biology hinge on whether they canbe used to improve the efficiency of chimeric rAAV/HBoV vectorproduction. The ability of a new packaging system to improve chimericrAAV/HBoV vector production was tested. Two series of HBoV1 capsidexpression constructs were generated and tested for their efficiency ofpseudopackaging the rAAV genome in HBoV capsid. One set of helpers wasNP1 protein expression dependent. They are the revised versions based onthe pHBoVNScap helper, but the expression of NS1, NS2, NS3 and NS4 geneis null and the endogenous P5 promoter is replaced with a strong humanCMV IE enhancer/promoter. This set of helpers increase vector productionby 6- to 12-fold with the yield of 3,000-6,000 DRP/cell (in crudelysate) or 1,500-2,500 DRP/cell (purified vector); the transductionactivity of the virus was similar to that produced with the pHBoVNSCaphelper. Another set of helpers was NP1 protein expression-independent.Mammalian cell expression vector pcDNA3.1 was used to express HBoV1capsid proteins from a synthetic HBoV1 cap cDNA (optVP1U or optVP1)without other HBoV1 sequence. optVP1U and optVP1 cDNAs were constructedwith a codon optimized capsid coding sequence as well as an alternativestart codon to initiate VP1 translation. In addition, the proximalpolyadenylation sites (pA)p in the 5′ end of capsid protein-encodingregion was removed. In optVP1U cDNA, the codon optimization isrestricted in the 5′ unique region of VP1 and VP2, whereas in optVP1,the codon optimization is through the entire encoding sequence of VP1,VP2 and VP3. The CMV enhancer/promoter sequence was used to express theoptVP1 cDNA, and HBoV1 VP1, VP2 and VP3 expression was as efficient asthe pHBoVNSCap helper and at a similar ratio of 1:1:10.60- to 80-foldhigher chimeric vector production was obtained from this set of NP1independent helpers. The vector production yields reach 15,000 to 40,000DRP/cell (in crude lysate) or >10,000 DRP/cell (purified vector).However, compromised transduction activity was observed. The potency ofthe vector produced from NP1 independent helpers is ⅙ to 1/10 of thoseproduced from the pHBoVNSCap or NP1 dependent helpers.

In summary, an improved rAAV/HBoV1 production system was developed thathad at least a 5 to 75-fold higher efficiency than the prototype system.The current production yield is good enough for us to conductpreclinical study of the rAAV/HBoV1 vector in CF ferret model. TherAAV/HBoV1 vector produced from the improved production system using theNP1-dependent helper was a cultured in polarized airway cells and inferrets in vivo. Although the transduction activity of the vectorproduced from the NP1-independent helper is compromised, its ability topackage rAAV/HBoV1 vector is superiorly high: up to 10-fold moreefficient than the NP1-dependent helper.

The invention will be further described by the following non-limitingexample.

Example Materials and Methods Plasmid Construction

The parent plasmid pHBoV1NSCap has been used to package rAAV2/HBoV1vector, which contains an incomplete HBoV1 genome (nt 97 to 5,395)without the intact left and right end hairpins (Yan et al., 2013). Allother plasmids based on the pHBoV1NSCap were constructed as follows, andthey are also diagrammed in the figures.

pHBoV1NSCap-Based Plasmids:

pVP2m1, pVP2m2, pVP2m3 and pVP2m4 were constructed by mutating nt3,422-3,427 of the HBoV1 sequence, as shown in FIG. 3A, in pHBoV1NSCap.pCMVNSCap-Based Plasmids:

pCMVNSCap was constructed by replacing the HBoV1 P5 promoter (nt 97-281)with the human cytomegalovirus immediate early enhancer/promotersequence (CMV) retrieved from the pcDNA3 vector (Invitrogen/ThermoFisher Scientific Inc. Grand Island, N.Y.) in pHBoV1NSCap.pCMVNSCapbGHpA was constructed by replacing the HBoV1 3′ untranslationalregion (UTR) (nt 5,168-5,395) with the bovine hormone genepolyadenylation signal (bGHpA) of the pcDNA3 in pCMVNSCap. Based on thepCMVNSCap, NS1, NP1, and both NS1 and NP1 encoding sequences were earlyterminated by introducing a stop codon (Huang et al., 2012), whichallowed construction of pCMVNS*Cap, pCMVNS(NP*)Cap and pCMVNS*(NP*)Cap,respectively.

VP cDNA Plasmids:

In pCMVNSCap, nt 282-3,091 of the HBoV1 sequence were deleted toconstruct pCMVCap1, and moved the VP1 open reading frame (ORF) directlyunder the CMV promoter to construct pCMVCap2. All D1-A1, D2-A2, andD3-A3 intron sequences were deleted to construct pCMVR6cDNA. D1-A2 andD3-A3 intron sequences were removed to construct pCMVR7cDNA. D1-A3intron sequence were removed to construct pCMVR8cDNA. These three cDNAconstructs coordinate with three VP-encoding mRNA transcripts that werepreviously identified, R6, R7, and R8 mRNAs, which are indicated in FIG.1A.

Intron-Replaced Plasmids:

The erythropoietin gene (Epo) intron 4 (Yan et al., 2000) was insertedbetween the D1 and A3 sites to construct pCMVCap3. Based on thepCMVNS*Cap plasmid, the D3-A3 intron sequence was deleted to constructpCMVNS*(ln3Δ)Cap. Based on the pCMVNS*(ln3Δ)Cap, the first and secondintrons were changed to Epo intron 1 and 4, respectively, to constructpCMVEpoln14(In3A)Cap. Additionally, all three introns in pCMVNSCap werechanged to Epo introns 1, 2, and 4, respectively, to constructpCMVEpoln124Cap.

(pA)p Knockout [m(pA)p] Constructs:

The potentially used five polyadenylation sites (PASs) and both theirupstream and downstream elements [m(pA)p], which span the coding regionfor the amino acids between methionines of the VP1 and VP3 ORFs, weresilently mutated through a Codon Optimization algorithm at IDT(Integrated DNA Technologies, Inc., Coralville, Iowa) (FIG. 2). Anoptimized Kozak sequence (GTT AAG ACG) was used to express VP1, andremained the GTG codon for VP2, in order to obtain an appropriate ratioof VP1 to VP2 and to VP3. To construct pCMVNS*(NP*)m(pA)pCap andpCMVNS*(ln3Δ)m(pA)pCap, the (pA)p sites in pCMVNS*(NP*)Cap andpCMVNS*(ln3Δ)Cap, respectively were mutated. In the pCMVR6-8cDNAconstructs, the (pA)p sites were mutated to make pCMVR6-8cDNAm(pA)p.

Other Plasmids:

pOpt-NP1 was constructed by inserting a codon optimized NP1 ORF, whichwas synthesized at IDT, into pLenti-CMV-IRES-GFP-WPRE vector (Chen etal., 2011) through XbaI and BamHI sites. pCI-mCherry-HA was constructedby inserting a C-terminal HA-tagged mCherry ORF into pCI vector(Promega, Madison, Wis.) through Xho I and Xba I sites. All nucleotidenumbers (nt) of the HBoV1 genome refer to the full-length HBoV1 genome(GenBank accession no.:JQ923422). Constructs were verified for HBoV1sequence and mutations by Sanger sequencing at MCLAB (South SanFrancisco, Calif.).

Cell Culture and Transfection

HEK293 cells (CRL-1573) were purchased from American Type CultureCollection (ATCC, Manassas, Va.), and were cultured in Dulbecco'smodified Eagle's medium (GE Healthcare Bio-Sciences, Piscataway, N.J.)with 10% fetal calf serum (Sigma-Aldrich, St. Louis, Mo.). Cells grownin 60-mm dishes were transfected with a total of 4 μg of plasmid DNAusing LipoD293 reagent (SignaGen Laboratories, Gaithersburg, Md.),following the manufacturer's instructions. The pLenti-CMV-IRES-GFP-WPREvector (Chen et al., 2011) was cotransfected into HEK293 cells to ensurethe same amount of plasmid DNA was transfected for each NP1complementation experiment. As a control for transfection, 0.4 μg ofpCI-mCherry-HA plasmid DNA was cotransfected into HEK293 cells.

Western Blotting

HEK293 cells were transfected with plasmids as indicated in each figure.Cells were harvested and lysed at 2 days post-transfection. Westernblotting was performed to analyze the lysates as described In Shen etal. (2015) using the specific antibodies described in each figure. Ratanti-HBoV1 VP, NP1, and NS1 C-terminus (NS1C) were produced previously(Shen et al., 2015). Anti-β-actin and anti-HA monoclonal antibodies werepurchased from Sigma-Aldrich.

RNA Isolation and Analyses

RNA Isolation:

Cytoplasmic RNA was purified from transfected cells following the QIAGENSupplementary Protocol using the RNeasy® Mini Kit (Qiagen, Valencia,Calif.). For the RNA samples used for RNA export examination, the samenumbers of cells were extracted for cytoplasmic RNA and total RNA usingthe RNeasy Mini Kit. Other total RNA samples were prepared using TrizolRegent (Invitrogen), according to the manufacturer's instructions.

Northern Blotting:

Five μg of cytoplasmic or total RNA samples was separated on 1.4%denaturation agarose gel, and was visualized using ethidium bromine (EB)staining. The stained 18S ribosome RNA (rRNA) bands served as loadingcontrol. Northern blot analysis was performed as described in Sun et al.(2009), using ³²P-labeled DNA probes as diagrammed in FIG. 1B. In somegels, an RNA ladder (Invitrogen) was used as a size marker (Qiu et al.,2002).

RNAse Protection Assay (RPA):

RPA was performed as described in Chen et al. (2010) and Sun et al.(2009). RPA probes were constructed by cloning the indicated sequence(FIG. 1B) into BamHI/HindIII-digested vector pGEM4Z (Promega). Theprotected bands by the indicated probe are diagrammed in FIG. 1B.

Quantification:

Images of both Northern blotting and RPA were processed using a GETyphoon FLA 9000 phosphor imager (GE Healthcare Bio-Sciences,Pittsburgh, Pa.). ImageQuant TL 8.1 software was used to quantify thebands on the images.

TABLE 1 rAAV2/HBoV Production from HBoV Packaging Helpers Left RelativeHBoV NS gene expression Production Sequence Promoter pA (pA)p NS1 NS2NS3 NS4 NP1 Yield Prototype Helper pHBoV1NScap YES P5 (pA)d YES YES YESYES YES YES 1   NP1 dependent helper pCMVNS1(−)Cap YES CMV IE (pA)d YESNO NO YES YES YES 5.3 +/− 1.0 pCMVHBoVNS1-3(−)cap YES CMV IE (pA)d YESNO NO NO YES YES 9.3 +/− 0.5 pCMVHBoVNS1-3(−)D'm YES CMV IE (pA)d YES NONO NO NO YES 10.7 +/− 3.5  NP1 independent, VP1 and VP2 5′ unique regioncodon optimized pCMV-R7optVP1U NO CMV IE (pA)d NO NO NO NO NO NO 1.8 +/−0.4 pCMV-NS1(−)NP1(−)optVP1U YES CMV IE (pA)d NO NO NO YES YES NO 11.7+/− 2.9  pCMV-NS1-3(−)NP1(−)optVP1U YES CMV IE (pA)d NO NO NO NO YES NO4.3 +/− 0.8 pcDNAoptVP1hyd NO CMV IE SV40 pA NO NO NO NO NO NO 9.5 NP1independent, VP1, VP2 and VP3 codon fully optimized pCDNAoptVP1 NO CMVIE SV40 pA NO NO NO NO NO NO 63.2 +/− 10.5 Left HBoV Sequence: about 2.4kb HBoV1 genome encodes the non-structural proteins P5: EndogenousPromoter in HBoV1 genome (pA)d: 3′ distal polyadenylation site in HBoVGenome (pA)p: proximal polyadenylation sites at center of HBoV genome(within coding sequence of 5′ unique sequence of VP1/VP2) CMV IE: humancytomegalovirus immediate early promoter/enhance SV40 pA: SV40 virussequence containing polyadenylation sites Relative production yield fromeach helper is normalized to that production of 400-600 DRP (DNAseldigestion resistant particle)/cell in the production system using theprototype helper, pHBoV1, and this yield is set to 1.

Results

Identification of a Non-Canonical Initiation Site that Encodes a NovelCapsid Protein VP2

Previously, a band of capsid protein (VP*) was detected whose size isbetween VP1 and “VP2” in HBoV1-infected human airway epithelium andHBoV1 plasmid-transfected HEK293 cells (Shen et al., 2015), as well asin the purified rAAV2/HBoV1 vector (Yan et al., 2013). However, itwasn't known whether this VP* band is a cleaved protein of the VP1 or anovel capsid protein translated from a non-canonical initiation site, aGCU codon of the alanine at the amino acid (aa) 92 of the VP1 ORF, whichhas been previously Identified in the expression of VP1 ORF in insectSf9 cells (Cecchini et al., 2009). Therefore, this initiation site wasexamined in the expression of HBoV1 capsid proteins in HEK293 cells.Four mutations were made in the GUG and GCU codons in pHBoV1NSCap (FIG.3A). Two mutants that bear mutations of the GUG codon, which encode thevaline of aa 91 of the VP1 ORF, drastically decreased expression of theintermediate band between VP1 and VP3 (FIG. 3B, lanes 1&2). Thus, it wasconfirmed this intermediate band (VP*) of the HBoV1 capsid proteins is anovel capsid protein of VP2 initiated at the GUG codon at nt 3,422 ofthe HBoV1 genome.

HBoV1 VP cDNA is Intrinsically Inefficient to Generate VP-Encoding mRNA

In the rAAV2/HBoV1 vector production system, a non-replicating HBoV1construct pHBoV1NSCap was used as a packaging plasmid (Yan et al.,2013), which expressed NS1-4 and NP1 (Shen et al., 2015), in addition tocapsid proteins. To identify a simple packaging plasmid that expressesonly capsid proteins, expressive HBoV1 capsid proteins was attemptedectopically with minimal HBoV1 sequence containing the VP ORFs. To thisend, six VP expression plasmids under the control of CMV promoter, asoutlined in FIG. 4A, were constructed, including three VP cDNAconstructs of R6, R7, and R8 mRNAs (FIG. 1A), and the other three VP ORFconstructs that contain various sequences of 5′UTR. Surprisingly, noneof them expressed capsid proteins in transfected HEK 293 cells, asdetected by Western blotting (FIG. 48, lanes 2-7). Next, Northernblotting was performed to analyze VP-encoding mRNA in cytoplasm using aVP mRNA-specific Cap probe (FIG. 1B). Only a low abundance of the VPmRNA was detected in the cytoplasmic RNA preparations of the cellstransfected with the three cDNA constructs and the pCMVCap3, which wasless than about 20% of the VP mRNA generated from the controlpHBoV1NSCap (FIGS. 4C&D, lanes 4-7). We detected almost no cytoplasmicVP mRNA from the cells transfected with pCMVCap1/2 (FIGS. 4C&D, lanes2&3).

Taken together, these results revealed that ectopic expression of theHBoV1 VP ORF is not sufficient to express capsid proteins, due to thepoor production of VP mRNA in the cytoplasm.

NP1 Protein Plays an Important Role in the Expression of Capsid Proteins

Next, it was investigated how the capsid proteins are expressed frompHBoV1NSCap. To explore systematically the effects of the cis-elementsof the viral genome and of the viral proteins in trans on the expressionof capsid proteins, five constructs were made, as shown in FIG. 5A.Exchange of either the P5 promoter with the CMV promoter or the 3′UTR tobGHpA did not affect the capsid protein in general (FIG. 5B, lanes 2&3vs. 1). Knockout of the NS1 and NS2 expression in pCMVNSCap (FIG. 5D,lane 4, NS1) did not diminish the level of capsid proteins (FIG. 5B,lane 4, VP). However, when NP1 expression was knocked out by earlytermination of the NP1 ORF, both NP1 knockout constructs pCMVNS(NP*)Capand pCMVNS*(NP*)Cap failed to express appreciable levels of capsidproteins (FIG. 5B, lanes 5&6). Next, the levels of the cytoplasmic VPmRNA In transfected cells were examined. Consistent with the capsidprotein expression, NP1 knockout nearly abolished VP mRNA in thecytoplasm (FIG. 5C, lanes 5&6).

Collectively, these results provided evidence that HBoV1 NP1 plays acritical role in the expression of capsid proteins, which is due to theincreased level of VP mRNA in cytoplasm, and that NS1 and NS2 proteinsin trans and the cis sequences of the P5 promoter and the 3′UTR are notessential to capsid protein expression.

NP1 Protein Facilitates Splicing of VP mRNA at the A3 Splice Acceptor

Next, it was investigated how NP1 regulates capsid protein expression.Since splicing at the A3 splice acceptor of the three introns isprerequisite to the production of VP mRNAs (FIG. 1A), we examined thefunction of NP1 in the splicing at the A3 splice acceptor. When NP1 wasknocked out, splicing at the A3 splice acceptor decreased by 78-fold(FIG. 6B, lanes 5 vs. 4), whereas the splicing of the first and secondintrons did not (FIG. 6B, lanes 2 vs. 1). Complementation of the NP1 Intrans restored 67% of the mRNA spliced at the A3 splice acceptor (FIG.6B, lane 6, and FIG. 6C). In parallel with the inefficient splicing atthe A3 splice acceptor, cytoplasmic VP mRNA was not detectable (FIG. 6D,lane 2), and capsid proteins were not expressed from pCMVNS*(NP)Cap(FIG. 6E, lane 2). However, complementation of the NP1 restored both theexpressions of VP mRNA and of capsid proteins (FIGS. 6D&E, lane 3).

The undetectable level of cytoplasmic mRNA from the NP1 knockout mutanttherefore was due to the inhibited production of VP mRNA in the nucleusand not due to the inefficient export of VP mRNA from the nucleus (FIG.6F, lanes 3&4). VP mRNA was exported from the nucleus to cytoplasmefficiently (FIGS. 6F&G, lanes 1 vs. 2 and 5 vs. 6). In addition, VPmRNA was quite stable in the cells for a period of 24 hours, asdetermined by the RNA stability assay using actinomycin D (FIG. 6H).

Taken together, these results confirmed that NP1 is required for thesplicing of HBoV1 mRNAs at the A3 splice acceptor, which determines thelevel of VP mRNA in the cytoplasm and, therefore, the production ofcapsid proteins.

NP1 Protein Activates VP mRNA Expression Independently of Splicing

To distinguish the function of NP1 in splicing and Internalpolyadenylation read-through of VP mRNA, three helper plasmids wereconstructed: 1) pCMVNS*(ln3Δ)Cap, in which the third Intron was removed(FIG. 7A, construct 1); 2) pCMVEpoln14(In3A)Cap, in which the first andsecond Introns were replaced with the Epo introns 1 and 4, respectively,and the third intron was also removed (FIG. 7A, construct 2); and 3)pCMVEpoln124Cap, in which all three introns were replaced with Epointrons (FIG. 7A, construct 3). Since NP1 protein was encoded by the ORF312 lying in the third intron sequence, all three constructs did notexpress NP1 (FIG. 7B, lanes 1-3). When NP1 was added back in trans, VPmRNA at approximately (about) 2.5 kb, which is likely the R6 mRNA thatreads through the (pA)p site and Is polyadenylated at the (pA)d site(FIG. 1A), increased by at least 5-fold (FIGS. 7C&D, VP mRNA), but the(pA)p mRNA at about 0.8 kb, which is spliced of all introns andpolyadenylated at the (pA)p site, either remained unchanged or wassignificantly decreased (FIGS. 7C&D, (pA)p).

As controls, NP1 did not alter splicing of the Introns 1 and 2 of themRNAs generated from pCMVNS*(ln3Δ)Cap (FIG. 7E, lanes 2&5) or altersplicing of the heterogeneous introns (Epo introns 1 and 4) of the mRNAsgenerated from pCMVEpoln14*(ln3Δ)Cap (FIG. 7E, lanes 3&6). It also didnot alter splicing of the three heterogeneous Epo introns (Epo Introns1, 2, and 4) of the mRNAs generated from pCMVEpoln124Cap (FIG. 7E, lanes4 vs. 7, and 8 vs. 9).

Thus, these results strongly suggested that the role of NP1 inIncreasing the read through of the (pA)p site (the level of VP mRNA) isindependent of splicing at the A3 splice acceptor and the interveningintron sequence. Since both the pCMVEpoln14(In3A)Cap and pCMVEpoln124Capconstructs did not contain any NS ORFs, the function of NP1 infacilitating the VP mRNA to read through the (pA)p site is alsoIndependent of NS1-4.

Furthermore, the impact of NP1 on the expression of VP mRNA from variousVP cDNA constructs was evaluated. NP1 enhanced VP mRNA expression fromthe all VP cDNA constructs (FIG. 8B, lanes 4-6 vs. 1-3). With NP1provided In trans, the level of VP mRNA was increased by 3.7-, 4-, and6-fold from the expression of R6, R7, and R8 VP cDNAs, respectively(FIGS. 8B&C, lanes 1 vs. 4, 2 vs. 5 and 3 vs. 6). Again, as a control,with NP1 provided in trans, the NS1 and NP1 knockout constructpCMVNS*(NP*)Cap expressed VP mRNA (at about 2.5 kb; R6 In FIG. 1A), at alevel of over 30 times more than that without NP1, whereas the level of(pA)p mRNA (at about 1.5 kb; R5s in FIG. 1A) did not significantlychange (FIG. 8B, lanes 7 vs. 8, and FIG. 8C). However, the increased VPmRNAs from the cDNA constructs by the NP1 were still not sufficient toexpress capsid proteins (FIG. 8D, lanes 4-6). Of note, with the NP1provided in trans, the level of (pA)p mRNA (at about 0.8 kb) generatedfrom the cDNA constructs was not significantly changed (FIG. 8C, (pA)pmRNA). These results suggested that the Increased read through VP mRNAis not due to the simple conversion of the (pA)p mRNA to VP mRNA.

Taken together, these results confirmed that NP1 protein facilitates VPmRNA to read through the (pA)p site, independent of any splicing events.

Knockout of the Polyadenylation Signals in the Center of the ViralGenome Compensates for the Requirement of NP1 Protein in the Expressionof Capsid Proteins

Next, the role of the (pA)p site in the blockage of the read-through ofVP mRNA and capsid protein expression was examined. First, mutations ofthe polyadenylation signal (PAS) AAUAAA site at nt 3,485 (Chen et al.,2010), as well as mutations of Its upstream and downstream regions thatoften regulate polyadenylation (Zarudnaya et al., 2003; Huang et al.,2012) were made, in pCMVNS*Cap. We failed to decrease the level of (pA)pmRNA or Increase the level of VP mRNA (data not shown). Since there area series of five PASs in the middle of the genome, the VP1 start-VP3start-encoding region, we made silent mutations of the entire VP1start-VP3 start-encoding sequence [m(pA)p], which covers al five PASs(FIG. 2). The constructs that bear the m(pA)p mutation were observed tono longer generate (pA)p mRNA (FIG. 9B, (pA)p, lanes 2, 3 and 7-9), butproduced much higher levels of VP mRNA (FIGS. 9B&C, VP mRNA, lanes 2, 3,and 7-9). In agreement with this finding, the m(pA)p mutation enabledcapsid protein expression in the absence of NP1 from the NSCap geneconstructs (FIG. 9D, lanes 2&3 vs. 10), as well as the VP cDNAconstructs (FIG. 9D, lanes 7-9 vs. 4-6).

Thus, the present results confirmed that internal polyadenylationprevents HBoV1 pre-mRNA from transcribing through the (pA)p site, whichcontrols the production of VP mRNA, and, therefore, prevents theexpression of capsid proteins. Of note, VP mRNA expressed from the NSCapconstructs migrated at the same position as the mRNA expressed from theR8cDNA did (FIG. 9B, lanes 1, 2 and 3 vs. 7), suggesting that R6 mRNA isthe key VP mRNA.

Discussion

The NP1 protein is a unique small non-structural protein expressed onlyby members of the genus Bocaparvovirus among parvoviruses (Chen et al.,2010; Sun et al., 2009; Lederman et al., 1984; Qiu et al., 2007). It isrequired for efficient replication of Bocaparvovirus DNA (Huang et al.,2012; Sun et al., 2009). The NP1 protein shares features among membersof Bocaparvovirus. Both the BPV1 and HBoV1 NP1 proteins can complementthe loss of NP1 during MVC DNA replication to some extents (Sun et al.,2009). HBoV1 NP1 protein could complement some functions of the minutevirus of mice (MVM) NS2 during an early phase of Infection (Mihaylov etal., 2014).

In a previous study, MVC NP1 was shown to play a role in regulatingcapsid protein expression by facilitating VP mRNA transcript to readthrough the Internal polyadenylation site (Sukhu et al., 2012). However,in that study, the function of the NP1 in enhancing splicing of VP mRNAat the A3 splice acceptor and in solely facilitating the read-through ofthe (pA)p site of VP mRNA (Sukhu et al., 2012) was not differentiated,since all VP mRNAs have to be spliced at the A3 splice acceptor (Dijkmanet al., 2009; Chen et al., 2010, Sun et al., 2009). In the presentstudy, it was demonstrated that NP1 plays a double role in controllingthe production of VP mRNA. First, NP1 is critical to the splicing of theVP mRNA at the A3 splice acceptor, which is essential to generate VPmRNA. Second, NP1 facilitates viral pre-mRNA to read through theInternal (pA)p site for the production of VP mRNA, independently of anysplicing events. More importantly, the function of NP1 in capsid proteinexpression is Independent of the other four non-structural proteins(NS1-4).

It was observed that when splicing is involved (from the constructs thatcontain introns), NP1 increased the level of VP mRNA on average to amuch greater extent than in the absence of splicing (from these cDNAconstructs) (FIGS. 7 vs. 8). This finding suggests that splicing boostsNP1-facilitated read-through of the (pA)p site. Of note, while NP1increases the read through transcript VP mRNA, (pA)p mRNA does notdecrease significantly in most cases, suggesting that the increasedread-through of transcripts (VP mRNA) is not merely a conversion of the(pA)p mRNA, but is likely an activation of transcription. Moreover,replacing the D3-A3 Intron with heterogeneous Epo intron 4 destroyed theNP1 protein dependence of the splicing at the A3 splice acceptor (FIG.7), suggesting that the role of NP1 in enhancing splicing of VP mRNA atthe A3 splice site is dependent on the intervening sequence of the thirdintron. As we know, during cellular mRNA processing, RNA transcription,splicing, and polyadenylation are all coupled (Bentley, 2014).Therefore, it was speculated that NP1 may target the transcriptioncomplex, which initiates at the P5 promoter to activate transcription,enhances splicing at the A3 splice site, and prevents internalpolyadenylation, since VP mRNA must be spliced at the A3 splice site andread through the (pA)p site. Therefore, the HBoV1 NP1 is the firstexample for a parvovirus nonstructural protein that has multiplefunctions in viral pre-mRNA processing.

The level of the VP mRNAs is not proportionally related to the level ofcapsid proteins (FIGS. 7 & 9). One interpretation could be that there isa minimal level of VP mRNA required for the translation of capsidproteins. However, in the cases of highly expressed VP mRNA from these(pA)p knockout constructs (FIG. 9), the higher level of VP mRNA did notexpress a higher level of capsid proteins. NP1 may play a role in thetranslation of VP mRNA. Without NP1 expression from the (pA)p knockoutconstructs, a higher level of VP1 mRNA is required to efficientlytranslate capsid proteins.

In this study, expression of the novel VP2 from a non-canonicaltranslation initiation site (GUG) ORF was confirmed. Importantly, simpleHBoV1 VP ORF constructs (pCMVR6-cDNAm(pA)p), were identified which donot express any NS proteins (NS1-4 and NP1), express HBoV1 capsidproteins VP1, VP2, and VP3 at a level and at a ratio (VP1:VP2 and VP3)similar to that of the packaging helper plasmid pHBoV1NSCap forrAAV2/HBoV1 vector production (Yan et al., 2013). In fact, pCMVR8cDNApackaged rAAV2/HBoV1 vector at an efficiency higher than that by thepHBoV1NSCap (data not shown). Thus, the cDNAm(pA)p constructs will aidin the optimization of rAAV2/HBoV1 vector production in HEK293 cellswithout Interference from any HBoV1 NS proteins.

In summary, to improve the packaging efficiency of the pHBoV1NSCap inHEK293 cells, the expression of the HBoV1 capsid proteins was studied.Expression of HBoV1 capsid proteins was found to be regulated by NP1,but not by NS1, NS2, NS3, and NS4. Without NP1, HBoV1 capsidprotein-encoding transcripts are expressed at a low level that is notsufficient for the expression of capsid proteins.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1. A method of preparing a chimeric virus comprising bocavirus capsidprotein (VP) and a recombinant adeno-associated (AAV) viral genome,comprising: a) providing a mutant bocavirus genome that when introducedto cells; i) does not express one or more of bocavirus NS1, NS2, NS3 orNS4; ii) expresses bocavirus VP1, VP2 and VP3; and iii) expressesbocavirus NP1; b) introducing the mutant bocavirus genome and a rAAVvector to cells which do not express one or more of bocavirus NS1, NS2,NS3 or NS4, thereby producing bocavirus VP, bocavirus NP1 and a rAAVgenome; and c) collecting chimeric rAAV/bocavirus.
 2. The method ofclaim 1 wherein the rAAV vector comprises an expression cassetteencoding a heterologous gene product.
 3. The method of claim 2 whereinthe gene product encodes a therapeutic protein.
 4. The method of claim 1wherein the rAAV genome is a rAAV-1, rAAV-2, rAAV-3, rAAV-4, rAAV-5,rAAV-6, rAAV-7, rAAV-8 or rAAV-9 genome.
 5. The method of claim 2wherein the gene product is a viral, bacterial, tumor, parasite, orfungal antigen.
 6. The method of claim 2 wherein the gene product iscystic fibrosis transmembrane conductance regulator, β-globin, γ-globin,tyrosine hydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C,C1 inhibitor gene, C1-INH gene, SERPING gene, erythropoietin, HBoVprotein, influenza virus protein, RSV protein, a neutralizing antibodyor an antigen binding fragment thereof, SARS virus protein, or acytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, or IL-6. 7-8.(canceled)
 9. A method of preparing a chimeric virus comprisingbocavirus capsid protein and a recombinant adeno-associated (AAV) viralgenome which method is optionally independent of bocavirus NP1,comprising: a) providing one or more vectors that express bocavirus VP1,VP2 and VP3 and express bocavirus NP1, but do not express one or more ofbocavirus NS1, NS2, NS3 or NS4 or, if the method is independent ofbocavirus NP1 providing a mutant bocavirus genome that when introducedto cells i) do not express any of, or expresses one or more of,bocavirus NS1, NS2, NS3 or NS4, and ii) expresses bocavirus VP1, VP2 andVP3, iii) do not express a functional bocavirus NP1, wherein the codingregion for the one or more of the VP1, VP2 or VP3 in the mutant vectoris codon optimized; b) expressing the one or more vectors in cells thatdo not express one or more of bocavirus NS1, NS2, NS3 or NS4 and thathave a rAAV vector of if the method is independent of bocavirus NP1,introducing the mutant bocavirus genome and a rAAV vector into cellsthat do not express bocavirus NP1 in trans, thereby producing bocavirusnonstructural proteins NS1, NS2, NS3 or NS4 capsid proteins and a rAAVgenome; and c) collecting chimeric rAAV/bocavirus virus. 10-11.(canceled)
 12. The method of claim 9 wherein in the method that isindependent of NP1 the rAAV vector comprises an expression cassetteencoding a heterologous gene product.
 13. The method of claim 12 whereinthe gene product encodes a therapeutic protein.
 14. The method of claim9 wherein the rAAV genome is a rAAV-1, rAAV-2, rAAV-3, rAAV-4, rAAV-5,rAAV-6, rAAV-7, rAAV-8 or rAAV-9 genome.
 15. The method of claim 9wherein the gene product is a viral, bacterial, tumor, parasite, orfungal antigen.
 16. The method of claim 9 wherein the gene product iscystic fibrosis transmembrane conductance regulator, β-globin, γ-globin,tyrosine hydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C,C1 inhibitor gene, C1-INH gene, SERPING gene erythropoietin, HBoVprotein, influenza virus protein, RSV protein, a neutralizing antibodyor an antigen binding fragment thereof, SARS virus protein, or acytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, or L-6.
 17. Themethod of claim 9 wherein the bocavirus is a human bocavirus.
 18. Themethod of claim 9 wherein in the method that is independent of bocavirusNP1 neither the cells nor the mutant genome provides for expression ofany of bocavirus NS1, NS2, NS3 or NS4.
 19. A method of preparing achimeric virus comprising bocavirus capsid protein and a recombinantadeno-associated (AAV) viral genome, which method is independent ofbocavirus NP1, comprising: a) providing one or more vectors that whenintroduced to cells i) do not express any of, or express one or more of,bocavirus NS1, NS2, NS3 or NS4, ii) express bocavirus VP1, VP2 and VP3,iii) do not express a functional bocavirus NP1, wherein the codingregion for the one or more bocavirus VP1, VP2 or VP3 is codon optimized;b) expressing the one or more vectors and a rAAV vector in cells that donot express NP1 in trans, thereby producing bocavirus nonstructuralproteins NS1, NS2, NS3 or NS4 capsid protein and a rAAV genome; and c)collecting chimeric rAAV/bocavirus.
 20. The method of claim 19 whereinthe one or more vectors are integrated into the genome of the cell. 21.The method of claim 19 wherein neither the cells nor the one or morevectors provides for expression of any of bocavirus NS1, NS2, NS3 orNS4.
 22. The method of claim 1 wherein the promoter that expressesbocavirus proteins is not a bocavirus promoter.
 23. An isolated mutantbocavirus genome that when introduced to cells i) does not express oneor more of bocavirus NS1, NS2, NS3 or NS4; ii) expresses bocavirus VP1,VP2 and VP3; and iii) expresses bocavirus NP1; or a) expresses one ormore of bocavirus NS1, NS2, NS3 or NS4; b) expresses bocavirus VP1, VP2and VP3; and c) does not express a functional bocavirus NP1; or A) doesnot express any of bocavirus NS1, NS2, NS3 or NS4; B) expressesbocavirus VP1, VP2 and VP3; and C) does not express a functionalbocavirus NP1. 24-25. (canceled)
 26. The mutant genome of claim 23wherein the promoter that expresses bocavirus proteins is not abocavirus promoter. 27-28. (canceled)